Identification of potential serum metabolic biomarkers for patient with keratoconus using untargeted metabolomics approach.

This study aimed to investigate the metabolite differences between patients with keratoconus and control subjects and identify potential serum biomarkers for keratoconus using a non-targeted metabolomics approach. Venous blood samples were obtained from patients with keratoconus (n = 20) as well as from age-, gender- and race-matched control subjects (n = 20). Metabolites extracted from serum were separated and analyzed by liquid chromatography/quadrupole time-of-flight mass spectrometer. Processing of raw data and analysis of the data files was performed using Agilent Mass Hunter Qualitative software. The identified metabolites were subjected to a principal component and hierarchical cluster analysis. Appropriate statistical tests were used to analyze the metabolomic profiling data. Together, the analysis revealed that the dehydroepiandrosterone sulfate from the steroidal hormone synthesis pathway was significantly upregulated in patients with keratoconus (p < 0.05). Also, a combination of eicosanoids from the arachidonic acid pathway, mainly prostaglandin F2α, prostaglandin A2, 16,16-dimethyl prostaglandin E2, and 5-hydroxyeicosatetraenoic acid were collectively up-regulated as a group in keratoconus patients (p < 0.05). On the other hand, glycerophospholipid PS(17:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)) was found to be significantly upregulated in the metabolomics profiles of control subjects (p < 0.05). The differently regulated metabolites provide insights into the pathophysiology of keratoconus and could potentially be used as biomarkers for keratoconus to aid in screening for individuals at risk hence, enabling early diagnosis and timely monitoring of disease.

Serum Inflammation Biomarkers in Patients with Keratoconus.

Purpose: To evaluate inflammation-sensitive parameters, namely the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), eosinophil count, erythrocyte distribution width (RDW), and CRP/albumin ratio in patients with keratoconus (KC).Methods: Thirty-five patients with KC and 30 healthy people (control group) were included in the study. Complete blood count measurements were performed in all subjects, and NLR, PLR and the CRP/albumin ratio were calculated.Results: NLR was determined as 2.01 ± 0.53 in patients with KC and 1.97 ± 0.41 in the control group. PLR was 146.20 ± 23.32 in the KC group and 121.45 ± 31.75 in the healthy group. The CRP/albumin ratio was determined as 0.33 ± 0.09 in the KC group and 0.29 ± 0.11 in the healthy group.Conclusion: Although no significant difference was found between the groups in terms of NLR, CRP/albumin ratio and RDW levels, PLR was significantly higher in the KC group.

Serum 25-Hydroxy Vitamin D, Vitamin B12, and Folic Acid Levels in Progressive and Nonprogressive Keratoconus.

PURPOSE: To evaluate the serum 25-hydroxy vitamin D, vitamin B12, and folic acid (FA) levels in progressive and nonprogressive patients with keratoconus (KCN). METHODS: Fifty-five patients with KCN (28 progressive and 27 nonprogressive) who were followed up for at least 12 months were recruited and separated into 2 groups according to corneal topographic progression criteria. Age- and sex-matched 30 healthy individuals formed the control group. Serum vitamin D, B12, FA, and Ca levels were compared among control, nonprogressive, and progressive groups. The effect of gender, age, vitamin D, B12, and FA parameters on KCN progression was investigated. RESULTS: Serum vitamin D levels were 12.77 ± 5.52, 11.78 ± 4.32, and 17.40 ± 5.12 ng/mL in nonprogressive, progressive, and control groups, respectively. The serum vitamin D levels in KCN groups (nonprogressive and progressive) were significantly lower than the control group (P = 0.002 and P < 0.001, respectively). There was no significant difference between all groups in serum vitamin B12, FA, and Ca levels (P > 0.05). Decreased vitamin D levels significantly increased nonprogressive KCN probability 1.23 times and progressive KCN probability 1.29 times more than the control group (P = 0.002, 95% confidence interval, 0.708-0.925; P < 0.001, 95% confidence interval, 0.668-0.888, respectively). CONCLUSIONS: Serum vitamin D levels were significantly decreased in both KCN groups. However, serum vitamin B12, FA, and Ca levels were similar in all groups. Serum vitamin D evaluation of patients with KCN at onset and follow-up examinations may help to predict the course of the disease.

Serum Vitamin D Levels in Patients with Keratoconus.

Purpose: To evaluate serum 25-hydroxyvitamin D levels in patients with keratoconus (KC) and its association with disease severity and to compare vitamin D levels with those of age- and sex-matched healthy individuals.Methods: This study included 100 patients with KC and 100 non-atopic healthy controls. Serum vitamin D levels were measured using liquid chromatography-tandem mass spectrometry and compared between the study groups. KC severity was graded as mild (K = 43.1-46.9 D), moderate (K = 47-50 D), and severe (K > 50 D). Vitamin D levels were classified as deficient (≤10 ng/mL), insufficient (11-20 ng/mL), and optimal (>20 ng/mL).Results: The mean serum vitamin D level was significantly lower in the KC group than in the control group (12.18 ± 5.05 ng/mL vs 15.18 ± 5.81 ng/mL; p < .001). Vitamin D deficiency (≤10 ng/mL) was observed in 54.0% of the KC patients and 34.0% of the controls (p = .011). Multivariable logistic regression analysis revealed that having deficient vitamin D level increased the presence of KC 2.9 fold (p = .021, 95% confidence interval, 1.17-6.95). Decreasing serum vitamin D levels were not significantly associated with increasing severity of KC (p = .311). Although the rate of patients with deficient vitamin D was the highest in the severe KC group, distribution of vitamin D levels were not significantly different among KC groups (p = .326).Conclusion: The patients with KC had lower serum vitamin D levels than those of age- and sex-matched healthy controls. The present study could be a valuable entry point for further researches investigating the potential link between vitamin D and KC.

Gonadotropins in Keratoconus: The Unexpected Suspects.

Keratoconus (KC) is the most common ectatic corneal disease with a significant visual acuity burden. The actual burden is intangible given that KC can disrupt daily activities (reading, driving, and various career paths). Despite decades of research and clinical studies, the etiology, onset, and pathobiology of KC remain a mystery. The purpose of this study was to investigate the role of gonadotropins in KC. We recruited 86 KC patients (63 males, 23 female), and 45 healthy controls (22 male, 23 female). Plasma samples were collected and analyzed using an enzyme-linked immunosorbent assay. Corneal stromal cells from KC and healthy controls, and human epithelial corneal cells, were also investigated for gonadotropin-related markers. Our results show significant alterations of LH/FSH in KCs, compared to healthy controls. Our data also reveals, for the first time, the existence of gonadotropins and their receptors in KC. Our study is the first to demonstrate the role of LH/FSH in KCs, and expand the list of organs known to express gonadotropins, or their receptors, to include the human cornea. Our findings suggest that the human cornea is capable of responding to gonadotropins, and propose an intriguing mechanism for the onset and/or progression of KC.

Association Between Advanced Keratoconus and Serum Levels of Zinc, Calcium, Magnesium, Iron, Copper, and Selenium.

PURPOSE: To investigate the possibility of an association between serum levels of zinc (Zn), calcium (Ca), magnesium (Mg), iron (Fe), copper (Cu), and selenium (Se) and advanced keratoconus (KCN). METHODS: In this study, 50 patients with advanced KCN and 50 control subjects were included. Plasma levels of Zn, Ca, Mg, Fe, Cu, and Se were compared between the groups. RESULTS: Mean ± SD age of the patients in the case and control groups was 29.3 ± 3.5 and 28.9 ± 5.9 years, respectively (P = 0.669). Among serum parameters, there were no statistically significant differences between the 2 groups in serum levels of Ca, Mg, and Fe (P > 0.05), whereas serum levels of Zn and Cu were significantly lower in the case group than the control group [65.92 ± 7.90 vs. 87.36 ± 13.64 µg/dL and 78.48 ± 11.96 vs. 116.34 ± 20.87 µg/dL, respectively (both P < 0.001)] as well as the serum level of Se [79.76 ± 19.16 vs. 87.41 ± 15.91 µg/dL (P = 0.032)]. CONCLUSIONS: The lower serum levels of Zn, Cu, and Se in patients with advanced KCN compared with healthy controls indicate the possible role of antioxidants in the etiopathogenesis of KCN, which points to early treatment of this progressive degenerative disease with supplementation therapy, rather than keratoplasty techniques.

Higher Expression of Toll-Like Receptors 2 and 4 in Blood Cells of Keratoconus Patiens.

Inflammation may play a significant role in Keratoconus (KC), but the implication of immunity on this inflammatory response is unknown. Therefore, our aim was to determine the expression levels of Toll-like receptors 2 (TLR2) and 4 (TLR4) in monocytes and neutrophils from patients with KC and control subjects for demonstrating the role of innate immunity in KC. We also study the correlation between TLR2/TLR4 expression and serum levels of proinflammatory markers (IL-1ß, IL-6, TNF-α, MMP-9 and NF-κB). Forty patients with bilateral KC (55% males; mean age; 33.1 ± 10.9 years) and 20 control subjects (55% males; mean age; 30.4 ± 7.6 years) were included in the study. Our results showed that mean expression of TLR2 and TLR4 in both neutrophils and monocytes was significantly higher in patients with KC compared to control subjects (all p < 0.0001). Furthermore, KC patients also showed higher serum levels of IL-1B, IL-6, TNF-α, MMP-9 (all p < 0.0001) and NF-κB (p = 0.036). In addition, we found a strong correlation between TLR2 expression in both monocytes and neutrophils (all p < 0.0001), and TLR4 in monocytes (all p < 0.05) with serum levels of IL-1B, IL-6, TNF-α and MMP-9. In conclusion, these findings suggest that TLRs may play an important role in the pathophysiology of KC.

Evaluation of Serum Superoxide Dismutase Activity, Malondialdehyde, and Zinc and Copper Levels in Patients With Keratoconus.

PURPOSE: The aim of this study was to evaluate the relationship between antioxidant superoxide dismutase (SOD) enzyme activity, malondialdehyde (MDA) as a lipid peroxidation marker, and some trace elements such as zinc (Zn) and copper (Cu) levels in patients with keratoconus. METHODS: A total of 58 patients with keratoconus and 53 control subjects with similar age and sex were evaluated in this study. The modified Krumeich keratoconus classification was used to divide the patients into 4 stages. Serum SOD activity, MDA, and zinc and copper levels were compared between the patient and control groups. RESULTS: The median serum SOD activity, MDA, and Zn and Cu levels were 27.2 (42.4-13.7) U/mL, 10.2 (11.9-8.5) nmol/mL, 87.9 (104.6-76.5) µmol/L, and 103.2 (117.9-90.3) µmol/L in the keratoconus group and 26.2 (32.5-14.4) U/mL, 8.8 (11.4-7.1) nmol/mL, 100.5 (121.1-81.8) µmol/L, and 98.4 (120.3-83.4) µmol/L in the control group, respectively. There was a statistically significant difference between the MDA and Zn levels of the keratoconus group and control subjects but not between the respective SOD activities or Cu levels (P = 0.016, P = 0.031, P = 0.440, and P = 0.376, respectively). We found no significant difference between the keratoconus group stages for serum SOD activity, serum MDA, and Zn and Cu levels (P > 0.05), and there was also no significant correlation between the keratoconus group stages and serum SOD activity, serum MDA, and Zn and Cu levels (P > 0.05). CONCLUSIONS: There is imbalance in the systemic oxidant/antioxidant status where Zn deficiency also plays a role in patients with keratoconus.

Distribution of ABO and Rh Blood Groups in Patients With Keratoconus: A Case-Control Study.

UNLABELLED: Association of keratoconus (KC) with genetic predisposition and environmental factors has been well documented. However, no single study has investigated the possible relationship between ABO and Rh blood groups and KC. METHODS: A case-control study was designed in a university hospital enrolling 214 patients with KC in the case group and equal number of age- and sex-matched healthy subjects in the control group. Primary characteristics, ABO blood group, and Rh factors were compared between the two groups. Topographic findings of KC eyes and the severity of the diseases were investigated according to the distribution of the blood groups. RESULTS: Blood group O and Rh(+) phenotype were most frequent in both groups. There was no significant difference between the two groups in terms of ABO blood groups or Rh factors. Mean keratometery (K), central corneal thickness, thinnest corneal thickness, flat K, steep K, sphere and cylinder, spherical equivalent, and uncorrected visual acuity were all similar between ABO blood groups and Rh(+) and Rh(-) groups. However, the best spectacle-corrected visual acuity (BCVA) had the highest value in AB blood group (0.35 ± 0.22 logMAR, P=0.005). Moreover, the blood group AB revealed the highest frequency for grade 3 KC, followed by grades 1, 2, and 4 (P=0.003). CONCLUSION: We observed no significant excess of any particular blood group among KC cases compared with healthy subjects. Except BCVA, none of the keratometric or topographic findings was significantly different between blood groups.

Increased systemic oxidative stress in patients with keratoconus.

PURPOSE: To establish the effect of systemic oxidative stress on the pathogenesis of keratoconus by measuring serum total oxidant status (TOS) and total antioxidant status (TAS) in patients with keratoconus. METHODS: Twenty-five patients with keratoconus (keratoconus group) and 25 age-sex-matched healthy subjects (control group) were enrolled in the study. Exclusion criteria were smoking habit, history of any other corneal pathology, systemic disease or inflammation, and current antioxidant or anti-inflammatory therapies. All participants underwent a detailed ophthalmological examination and corneal topography. Serum samples were obtained from all participants. Oxidative stress markers (TAS and TOS) were measured using a commercial kit and oxidative stress index (OSI) was calculated. RESULTS: The study comprised 25 patients with keratoconus (mean age of 26.4±1.7 years) and 25 healthy control subjects (mean age of 26.6±1.7 years) (P>0.05). The serum TOS and OSI values were significantly higher in patients with keratoconus compared with those of the controls (P=0.036 and 0.037, respectively). However, serum TAS did not show significant difference between the keratoconus and control groups (P=0.497). CONCLUSIONS: The higher levels of serum oxidant status and OSI in patients with keratoconus suggest that systemic oxidative stress might be involved in the pathogenesis of keratoconus.

The relation between keratoconus and plasma levels of MMP-2, zinc, and SOD.

PURPOSE: To evaluate the levels of matrix metalloproteinase-2 (MMP-2), superoxide dismutase (SOD), and zinc in plasma taken from patients with keratoconus and to investigate the likely association between these factors and keratoconus. METHODS: A total of 36 patients with keratoconus and 40 control subjects at the Department of Ophthalmology, Faculty of Medicine, Gaziosmanpasa University, were included in the study. Plasma levels of zinc were determined with atomic absorption spectrometry for all the subjects. Measurements of plasma MMP-2 levels were performed by the enzyme-linked immunosorbent assay method. Total plasma (Cu/Zn and Mn) SOD activity was also determined photometrically. RESULTS: The plasma concentrations of zinc and MMP-2 were significantly lower in patients with keratoconus than in the healthy controls (P < 0.001). Total plasma SOD levels were significantly higher in patients with keratoconus than in the healthy controls (P < 0.001). CONCLUSIONS: We detected reduced plasma levels of zinc and MMP-2, and enhanced plasma levels of SOD in patients with keratoconus compared with the healthy subjects. The data presented provide insight into the potential role these molecules may play in the etiopathogenesis of this disease.

Association of polymorphisms in the hepatocyte growth factor gene promoter with keratoconus.

PURPOSE: Keratoconus is a progressive disorder of the cornea that can lead to severe visual impairment or blindness. Although several genomic regions have been linked to rare familial forms of keratoconus, no genes have yet been definitively identified for common forms of the disease. METHODS: Two genome-wide association scans were undertaken in parallel. The first used pooled DNA from an Australian cohort, followed by typing of top-ranked single-nucleotide polymorphisms (SNPs) in individual DNA samples. The second was conducted in individually genotyped patients, and controls from the USA. Tag SNPs around the hepatocyte growth factor (HGF) gene were typed in three additional replication cohorts. Serum levels of HGF protein in normal individuals were assessed with ELISA and correlated with genotype. RESULTS: The only SNP observed to be associated in both the pooled discovery and primary replication cohort was rs1014091, located upstream of the HGF gene. The nearby SNP rs3735520 was found to be associated in the individually typed discovery cohort (P = 6.1 × 10(-7)). Genotyping of tag SNPs around HGF revealed association at rs3735520 and rs17501108/rs1014091 in four of the five cohorts. Meta-analysis of all five datasets together yielded suggestive P values for rs3735520 (P = 9.9 × 10(-7)) and rs17501108 (P = 9.9 × 10(-5)). In addition, SNP rs3735520 was found to be associated with serum HGF level in normal individuals (P = 0.036). CONCLUSIONS: Taken together, these results implicate genetic variation at the HGF locus with keratoconus susceptibility.

[The immunity of patients with keratoconus]. / Osobennosti immuniteta u bolnykh keratokonusom.

Cellular and humoral immunity parameters were studied in patients with keratoconus at different stages, aged 14 to 49 years. The results showed the immune homeostasis to be impaired in patients with local involvement of the cornea. At a total system's level the patients developed shifts in the relative content of T-lymphocytes and their subpopulations, dysimmunoglubulinemia with increased levels of immunoglobulins of the primary immune response in the blood serum, high levels of the C3 and C4 components with a reduction of the total hemolytic activity of the complement, increased levels of NBT-positive and phagocytic cells, and a decrease of the activity of serum lysozyme.

Keratoconus, magnesium deficiency, type A behavior, and allergy.

The etiology of keratoconus is still unknown. This project was designed to study, in a group of keratoconic patients and a control group, the following: clinical, endocrinological, immunological, psychological, and ophthalmological factors. We found mean serum magnesium deficiency and type A behavior to be significantly more common in keratoconic patients than in a control group of patients. In addition changes in gluco-mineral corticoids, changes in glucose metabolism, edema of allergic origin, and genetic factors may collaborate in the development of keratoconus. All these factors could affect the osmotic mechanism of the cornea: Na-K and/or Ca ATPase, the collagen structure by alteration of the adenylate cyclase activity, and other mechanisms. This study suggests an association between keratoconus, magnesium depletion, and type A behavior, which together constitute a new clinical syndrome and confirm an association between keratoconus and atopy.

Genetics in keratoconus: A, B, O blood groups.

A, B, O blood group frequencies and Rh factors were studied in 69 white keratoconus patients and compared with 1,465 healthy white individuals. We did not detect a significant excess of any particular blood group among keratoconus patients. It was concluded that blood groups were not associated with keratoconus.