Advancing Personalized Medicine by Analytical Means: Selection of Three Metabolites That Allows Discrimination between Glaucoma, Diabetes, and Controls.

This paper aimed at devising an intelligence-based method to select compounds that can distinguish between open-angle glaucoma patients, type 2 diabetes patients, and healthy controls. Taking the concentration of 188 compounds measured in the aqueous humour (AH) of patients and controls, linear discriminant analysis (LDA) was used to identify the right combination of compounds that could lead to accurate diagnosis. All possibilities, using the leave-one-out approach, were considered through ad hoc programming and in silico massive data production and statistical analysis. Our proof of concept led to the selection of four molecules: acetyl-ornithine (Ac-Orn), C3 acyl-carnitine (C3), diacyl C42:6 phosphatidylcholine (PC aa C42:6), and C3-DC (C4-OH) acyl-carnitine (C3-DC (C4-OH)) that, taken in combination, would lead to a 95% discriminative success. 100% success was obtained with a non-linear combination of the concentration of three of these four compounds. By discarding younger controls to adjust by age, results were similar although one control was misclassified as a diabetes patient. Methods based on the consideration of individual clinical chemical parameters have limitations in the ability to make a reliable diagnosis, stratify patients, and assess disease progression. Leveraging human AH metabolomic data, we developed a procedure that selects a minimal number of metabolites (3-5) and designs algorithms that maximize the overall accuracy evaluating both positive predictive (PPV) and negative predictive (NPV) values. Our approach of simultaneously considering the levels of a few metabolites can be extended to any other body fluid and has potential to advance precision medicine. Artificial intelligence is expected to use algorithms that use the concentration of three to five molecules to correctly diagnose diseases, also allowing stratification of patients and evaluation of disease progression. In addition, this significant advance shifts focus from a single-molecule biomarker approach to that of an appropriate combination of metabolites.

A metabolomics study in aqueous humor discloses altered arginine metabolism in Parkinson's disease.

BACKGROUND: The lack of accessible and informative biomarkers results in a delayed diagnosis of Parkinson's disease (PD), whose symptoms appear when a significant number of dopaminergic neurons have already disappeared. The retina, a historically overlooked part of the central nervous system (CNS), has gained recent attention. It has been discovered that the composition of cerebrospinal fluid influences the aqueous humor composition through microfluidic circulation. In addition, alterations found in the brain of patients with PD have a correlate in the retina. This new paradigm highlights the potential of the aqueous humor as a sample for identifying differentially concentrated metabolites that could, eventually, become biomarkers if also found altered in blood or CSF of patients. In this research we aim at analyzing the composition of the aqueous humor from healthy controls and PD patients. METHODS: A targeted metabolomics approach with concentration determination by mass spectrometry was used. Statistical methods including principal component analysis and linear discriminants were used to select differentially concentrated metabolites that allow distinguishing patients from controls. RESULTS: In this first metabolomics study in the aqueous humor of PD patients, elevated levels of 16 compounds were found; molecules differentially concentrated grouped into biogenic amines, amino acids, and acylcarnitines. A biogenic amine, putrescine, alone could be a metabolite capable of differentiating between PD and control samples. The altered levels of the metabolites were correlated, suggesting that the elevations stem from a common mechanism involving arginine metabolism. CONCLUSIONS: A combination of three metabolites, putrescine, tyrosine, and carnitine was able to correctly classify healthy participants from PD patients. Altered metabolite levels suggest altered arginine metabolism. The pattern of metabolomic disturbances was not due to the levodopa-based dopamine replacement medication because one of the patients was not yet taking levodopa but a dopamine receptor agonist.

New Biomarker Combination Related to Oxidative Stress and Inflammation in Primary Open-Angle Glaucoma.

Glaucoma is a multifactorial neurodegenerative disease and the second leading cause of blindness. Detection of clinically relevant biomarkers would aid better diagnoses and monitoring during treatment. In glaucoma, the protein composition of aqueous humor (AH) is relevant for the discovery of biomarkers. This study analyzes AH protein concentrations of putative biomarkers in patients with primary open-angle glaucoma (POAG) compared to a control group. Biomarkers were selected from known oxidative-stress and inflammatory pathways. Osteopontin (OPN), matrix metalloproteinase 9 (MMP-9), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), and interleukin-10 (IL-10) were measured using the ELISA technique. Thirty-two patients were recruited to the study, including sixteen control and sixteen glaucoma patients. The glaucoma group consisted of patients diagnosed with glaucoma. In both groups, the aqueous humor sample was obtained during cataract surgery. A significant increase in OPN, MMP-9, TNF-alpha, and IL-10 was observed in the POAG aqueous humor, compared to the control group (p < 0.05). Of note, the AH of POAG patients contained 5.6 ± 1.2-fold more OPN compared to that of control patients. Different expression profiles of oxidative stress-related and inflammatory biomarkers were observed between patients with POAG and controls. This confirms the reported involvement of inflammatory and oxidative stress pathways in POAG pathophysiology. In the future, several, targeted AH proteins may be used to generate a potential biomarker expression profile of this disease, aiding diagnoses and disease progression monitoring. This approach highlights the importance of biomarkers in the future. Biomarkers provide a way to measure disease progression and response to treatment. In the future, biomarkers will play a more critical role in the toolkit of ophthalmology healthcare professionals as the field moves towards personalized medicine and precision healthcare.

Biogenic Amine Levels Markedly Increase in the Aqueous Humor of Individuals with Controlled Type 2 Diabetes.

The composition of the aqueous humor of patients with type 2 diabetes is relevant to understanding the underlying causes of eye-related comorbidities. Information on the composition of aqueous humor in healthy subjects is limited due to the lack of adequate controls. To carry out a metabolomics study, 31 samples of aqueous humor from healthy subjects without ocular pathology, submitted to refractive surgery and seven samples from patients with type 2 diabetes without signs of ocular pathology related to diabetes were used. The level of 25 molecules was significantly (p < 0.001) altered in the aqueous humor of the patient group. The concentration of a single molecule, N-acetylornithine, makes it possible to discriminate between control and diabetes (sensitivity and specificity equal to 1). In addition, receptor operating characteristic curve and principal component analysis for the above-mentioned six molecules yielded significantly (p < 0.001) altered in the aqueous humor of the patient group. In addition, receptor operating characteristic curve and principal component analysis for six compounds yielded cut-off values and remarkable sensitivity, specificity, and segregation ability. The altered level of N-acetylornithine may be due to an increased amount of acetate in diabetes. It is of interest to further investigate whether this alteration is related to the pathogenesis of the disease. The increase in the amino form of pyruvate, alanine, in diabetes is also relevant because it could be a means of reducing the formation of lactate from pyruvate.

Targeted Metabolomics Shows That the Level of Glutamine, Kynurenine, Acyl-Carnitines and Lysophosphatidylcholines Is Significantly Increased in the Aqueous Humor of Glaucoma Patients.

The composition of the aqueous humor of patients with glaucoma is relevant to understand the underlying causes of the pathology. Information on the concentration of metabolites and small molecules in the aqueous humor of healthy subjects is limited. Among the causes of the limitations is the lack of healthy controls since, until recently, they were not surgically intervened; therefore, the aqueous humor of patients operated for cataract was used as a reference. Sixteen aqueous humor samples from healthy subjects undergoing refractive surgery and eight samples from glaucoma patients were used to assess the concentration of 188 compounds using chromatography and mass spectrometry. The concentration of 80 of the 188 was found to be reliable, allowing comparison of data from the two groups (glaucoma and control). The pattern found in the controls is similar to, but not the same as, that reported using samples from "controls" undergoing cataract surgery. Comparing data from glaucoma patients and healthy subjects, 57 of the 80 compounds were significantly (p < 0.05) altered in the aqueous humor. Kynurenine and glutamine, but not glutamate, were significantly increased in the glaucoma samples. Furthermore, 10 compounds were selected considering a statistical score of p < 0.0001 and the degree of change of more than double or less than half. The level of C10 (decanoyl)-carnitine decreased, while the concentration of spermidine and various acyl-carnitines and lysophosphatidylcholines increased in glaucoma. Principal component analysis showed complete segregation of controls and cases using the data for the 10 selected compounds. The receiver operating characteristic curve these 10 compounds and for glutamine allowed finding cut-off values and significant sensitivity and specificity scores. The concentration of small metabolites in the aqueous humor of glaucoma patients is altered even when they take medication and are well controlled. The imbalance affects membrane components, especially those of the mitochondria, suggesting that mitochondrial abnormalities are a cause or consequence of glaucoma. The increase in glutamine in glaucoma is also relevant because it could be a means of keeping the concentration of glutamate under control, thus avoiding its potential to induce the death of neurons and retinal cells. Equally notable was the increase in kynurenine, which is essential in the metabolism of nicotine adenine dinucleotides.

Melatonin counteracts oxidative damage in lens by regulation of Nrf2 and NLRP3 inflammasome activity.

Oxidative stress, generated because of an imbalance between reactive oxygen species (ROS) generation and elimination, is associated with lens damage and cataract progression. ROS generation is known to activate NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain-cointaining 3) inflammasome, and is believed to be an important link between oxidative stress and inflammation, that is also related to cataract development. Potential oxidative hazard to the lens by white light-emitting diode (LED) light, a source of illumination commonly used nowadays, has been suggested, although available information is limited. In this work, we evaluated the cytotoxicity induced by hydrogen peroxide (an oxidative stressor agent) and white LED light in lens epithelial cells as well as melatonin ability to counteract the effects induced by them. Melatonin is a neurohormone secreted by different ocular structures that could be useful to alleviate oxidative damage induced by different oxidative stressors in lens. Particularly, the modulation of Nrf2 (nuclear erythroid 2-related factor)/Keap 1 (Kelch-like ECH-associated protein 1), an essential oxidative stress regulator, and NLRP3 activity by melatonin was evaluated in lens epithelial cells. ROS levels rose after white LED light exposure and cell viability was reduced after challenge with oxidative stressor agents. Melatonin prevented cell death triggered by hydrogen peroxide and white LED light, precluded ROS generation induced by white LED light and promoted antioxidant lens capacity through upregulation of Nrf2 protein levels and SOD activity. NLRP3, caspase-1 and IL1-ß expression significantly increased in human lens cells exposed to H2O2 or irradiated with white LED light. Activation of NLRP3 inflammasome triggered by oxidative stressors was also abrogated by melatonin. Attenuation of inflammatory and cytotoxic effects induced by oxidative stressors provided by melatonin in lens indicate the interest of this molecule as a potential therapeutic agent for cataract prevention/management.

Modulation of aqueous humor melatonin levels by yellow-filter and its protective effect on lens.

Melatonin is mainly secreted by the pineal gland, and it is also produced by various ocular structures such as the lens. It has been recently demonstrated that melatonin ocular synthesis can be induced by blocking the blue component of white light by means of filters. Melatonin exhibits antioxidant properties that can be useful to face light-induced oxidative stress as well as oxidative events associated to ocular pathologies like cataracts. Moreover, as oxidative stress is a main event in cataract development, changes in melatonin levels could happen and be relevant in the progression of this pathology, a subject that remains uncertain. The goal of this work was to analyze the ability of a short wavelength light blocking (yellow) filter to modulate endogenous melatonin concentration and the antioxidant and cytoprotective actions induced by yellow filter's use in lens. Furthermore, we evaluated the potential changes in aqueous humor melatonin concentration from patients with cataracts. In human lens epithelial cells, white light-emitting diode (LED) light challenge reduced melatonin secretion, protein levels of the enzymes involved in melatonin synthesis (hydroxyindole-O-methyltransferase and unphosphorylated and phosphorylated forms of arylalkylamine N-acetyltransferase) and cell viability whereas increased reactive oxygen species production. Yellow filter exposure precluded melatonin secretion reduction and protected cells from oxidative damage. Consistent with cataract patient's results, significantly lower levels of melatonin were observed in aqueous humor of alloxan-induced diabetic cataract rabbits as compared to those of control rabbits. In contrast, aqueous humor melatonin levels of diabetic cataract animals maintaining in cages covered with a yellow filter resembled control values. This recovery seems to be mediated by the induction of melatonin biosynthetic enzymes protein expression. Yellow filter also preserved Nrf2 lens protein expression and superoxide dismutase protein levels and activity in diabetic animals. Modulation of endogenous ocular melatonin concentration using blocking filters might be a promising approach to prevent premature lens opacification.

Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension.

BACKGROUND: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3R and either MT1 (MT1R) or MT2 (MT2R) melatonin receptors. Heteromerization led to the blockade of D3R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. CONCLUSIONS: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3R-MT1R and D3R-MT1R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

Adreno-melatonin receptor complexes control ion homeostasis and intraocular pressure - their disruption contributes to hypertensive glaucoma.

BACKGROUND AND PURPOSE: Often, glaucoma presents with elevated eye hydrostatic pressure, which is regulated by endogenous melatonin. Phenylephrine increases cytoplasmic [Ca2+ ], via α1 -adrenoceptor activation, that is detrimental in glaucoma. The aims of this study were (a) to elucidate the role of melatonin receptors in humour production and intraocular pressure (IOP) maintenance and (b) to identify glaucoma-relevant melatonin-adrenoceptor interactions. EXPERIMENTAL APPROACH: Biophysical and proximity ligation assays were performed to identify interactions in heterologous expression systems, in cell lines and in human eyes. Gs /Gi /Gq signalling was investigated in these systems and in cells producing aqueous humour. IOP was determined in a mice model of glaucoma. Retinography and topically pharmacological treatment were performed in control and in glaucomatous mice. KEY RESULTS: α1 -adreno- and melatonin receptors form functional complexes in which the C-terminal tail of the adrenoceptor plays a role. Remarkably, activation of α1 -adrenoceptors in these complexes did not lead to cytosolic Ca2+ increases, suggesting Gs instead of Gq coupling is involved. The number of these complexes significantly decreased in models of glaucoma and, importantly, in human samples from glaucoma patients. This has led to hypothesize that melatonin, a hypotensive agent, plus blockade of α1 -adrenoceptors could normalize pressure in glaucoma. Remarkably, co-instillation of melatonin and prazosin, an α1 -adrenoceptor antagonist, resulted in long-term decreases in IOP in a well-established animal model of glaucoma. CONCLUSIONS AND IMPLICATIONS: The findings are instrumental to understand the physiological function of melatonin in the eye and its potential to address eye pathologies by targeting melatonin receptors and their complexes.

Effect of nutritional supplement based on melatonin on the intraocular pressure in normotensive subjects.

PURPOSE: To evaluate the effect of a new nutritional supplement based on melatonin on the intraocular pressure (IOP) in normotensive subjects. PATIENTS AND METHODS: A short-term prospective study was designed. Sixty-seven normotensive subjects were recruited. Patients were divided into two groups. The daily group (DG) (n = 18) was instructed to take the supplement between 22:00 and 23:00 (before sleeping) for 3 consecutive days. IOP was measured from 10.00 to 11.00 am the day before treatment and during the 3 days of experiment. The acute group (AG) (n = 49) was instructed to take the supplement after the second measure (11.00) of the second day. IOP was measured 1 h and just before the intake of the supplement and 1 and 2 h after. All measurements in this group were taken 1 day before without any supplement (control) and the day of experiment. RESULTS: The DG group showed a significant decrease in IOP after supplement intake in all days of experiment, from 14.9 ± 3.4 mm Hg to 13.8 ± 2.9 mm Hg after 3 days of experiment (p value < 0.001). For AG, IOP did not change during the control day; however, a reduction of 1 mm Hg was found 2 and 3 h after supplement intake, from 15.7 ± 2.5 mm Hg to 14.7 ± 2.5 mm Hg and 15.1 ± 2.7 mm Hg, respectively, being statistically significant (p value < 0.001). CONCLUSION: The supplement based on melatonin was able to reduce the IOP in normotensive subjects after 2 h of intake. Moreover, the daily intake showed a reduction in IOP during the 3 days of experiment.

TRPV4 Stimulation Induced Melatonin Secretion by Increasing Arylalkymine N-acetyltransferase (AANAT) Protein Level.

Melatonin is a molecule which has gained a great deal of interest in many areas of science; its synthesis was classically known to be in the pineal gland. However, many organs synthesize melatonin, such as several ocular structures. Melatonin is known to participate in many functions apart from its main action regulating the circadian rhythm. It is synthesized from serotonin in two steps, with a rate-limiting step carried out by arylalkymine N-acetyltransferase (AANAT). In this report, the role of TRPV4 channel present in human ciliary body epithelial cells in AANAT production was studied. Several experiments were undertaken to verify the adequate time to reach the maximal effect by using the TRPV4 agonist GSK1016790A, together with a dose-response study. An increase of 2.4 folds in AANAT was seen after 18 h of incubation with 10 nM of GSK1016790A (p < 0.001, n = 6). This increment was verified by antagonist assays. In summary, AANAT levels and therefore melatonin synthesis change after TRPV4 channel stimulation. Using this cell model together with human ciliary body tissue it is possible to suggest that AANAT plays an important role in pathologies related to intraocular pressure.

Elevated intraocular pressure increases melatonin levels in the aqueous humour.

PURPOSE: To study the levels of melatonin in the aqueous humour of normotensive and hypertensive intraocular pressure (IOP) patients and to compare them to an animal model of glaucoma. METHODS: A total of 37 eyes of 37 patients who underwent cataract surgery were included in the study and were divided into normotensive patients, with IOP below 21 mmHg (n = 23), and hypertensive patients, with IOP > 21 mmHg (n = 14). Glaucomatous DBA/2J (n = 6) and control C57BL/6J (n = 6) mice presenting 3 and 12 months of age for each strain were also used. Human and mice aqueous humours were aspirated using a 30-gauge Rycroft cannula on a tuberculin syringe and further processed to quantify melatonin by high-performance liquid chromatography analysis. RESULTS: Melatonin levels in normotensive patients (IOP below 21 mmHg) presented values as medians (first quartile; third quartile) of 14.62 (5.38;37.99) ng/ml (n = 23), while hypertensive patients (IOP above 21 mmHg) showed melatonin concentrations of 46.63 (10.28; 167.28) ng/ml (n = 14; p < 0.039). Glaucoma mice presented melatonin values of 0.37 (0.34; 0.59) ng/ml (at 3 months of age, before the pathology starts), which increased to 1.55 (0.94; 1.88) ng/ml (at 12 months of age, when the pathology is fully developed and IOP is maximum; n = 6, p < 0.001). Control mice did not significantly modified melatonin concentrations between 3 and 12 months of age. CONCLUSION: Patients with high IOP present increased concentrations of melatonin in their aqueous humour compared to normotensive patients. This has been confirmed in a glaucomatous animal model in which it has been possible to see a correlation between the development of the pathology, with an increase in IOP, and a concomitant elevation of melatonin in the aqueous humour.

Comparison of Intraocular Pressure before and after Laser In Situ Keratomileusis Refractive Surgery Measured with Perkins Tonometry, Noncontact Tonometry, and Transpalpebral Tonometry.

Purpose. To compare the intraocular pressure (IOP) before and after Laser In Situ Keratomileusis (LASIK), measured by Diaton, Perkins, and noncontact air pulse tonometers. Methods. Fifty-seven patients with a mean age of 34.88 were scheduled for myopia LASIK treatment. Spherical equivalent refraction (SER), corneal curvature (K), and central corneal thickness (CCT) and superior corneal thickness (SCT) were obtained before and after LASIK surgery. IOP values before and after surgery were measured using Diaton, Perkins, and noncontact air pulse tonometers. Results. The IOP values before and after LASIK surgery using Perkins tonometer and air tonometers were statistically significant (p < 0.05). However, no significant differences were found (p > 0.05) for IOP values measured with Diaton tonometer. CCT decreases significantly after surgery (p < 0.05) but no statistical differences were found in SCT (p = 0.08). Correlations between pre- and postsurgery were found for all tonometers used, with p = 0.001 and r = 0.434 for the air pulse tonometer, p = 0.008 and r = 0.355 for Perkins, and p < 0.001 and r = 0.637 for Diaton. Conclusion. Transpalpebral tonometry may be useful for measuring postsurgery IOP after myopic LASIK ablation because this technique is not influenced by the treatment.

Diadenosine polyphosphates after laser in situ keratomileusis and photorefractive keratectomy refractive techniques.

PURPOSE: To study the concentrations of diadenosine polyphosphates in the ocular surface after PRK and LASIK. METHODS: Sixty-one patients (30 males and 31 females) with ages ranging from 20 to 63 (34.04 ± 9.13 years) were recruited in Balear Institute of Ophthalmology, Palma de Mallorca, Spain. LASIK was performed in 92 eyes of 46 patients and PRK in 25 eyes of 15 patients. Variations in the levels of diadenosine polyphosphate (Ap4 A and Ap5 A), Schirmer I (Jones test), TBUT, corneal staining together with the Dry Eye Questionnaire to evaluate discomfort and dryness were studied. All tests were performed at the preoperative visit and at 1-day, 2-week, 1-month and 3-month postoperative visits. RESULTS: Ap4 A showed a 5 and 3.5 fold increase at the 1-day visit for LASIK and PRK, respectively. LASIK patients continued having higher statistically significant concentrations (p = 0.01) all over the follow-up. Ap5 A showed no significant differences at any visit. Tear volume decreased during the 3 months in LASIK. The PRK cases had a normal volume at 1 month. TBUT in LASIK increased at the 1-day visit (p = 0,002) and decreased from the 2 weeks onwards and for the PRK, decreased by a 35% at the 1-day visit and kept reduced for a month. Discomfort only increased at the 1-day visit (p = 0.007). Dryness frequency was similar in all visits. CONCLUSIONS: Ap4 A levels only are increased in refractive surgery patients during the first day after the surgery. This increasing suggests that Ap4 A may help accelerating the healing process.

Impact and permanence of LASIK-induced structural changes in the cornea on pneumotonometric measurements: contributions of flap cutting and stromal ablation.

PURPOSE: To determine the factors that lead to changes in intraocular pressure (IOP) measurements after laser-assisted in situ keratomileusis (LASIK) and their long-term stability. PATIENTS AND METHODS: Five hundred twenty-two myopic eyes and 296 hyperopic eyes were enrolled in the study. Pneumotonometry was used to measure IOP once in the preoperative stage and twice in the postoperative stage-1 month after the operation and 1 year later. Ultrasonic pachymetry was used to determine preoperative and intraoperative corneal thicknesses and axial length of the eye, whereas optical pachymetry was used in the preoperative stage and 1 month after surgery. Corneal topography was used to determine the preoperative and postoperative mean curvature of the anterior surface of the cornea over 3 and 5-mm diameter regions. Comparative statistical analysis of the retrospective data series was performed. RESULTS: A highly significant reduction of IOP readings is found after LASIK for both myopic and hyperopic eyes. The reduction is stable 1 year after LASIK. In the case of myopic eyes, the reduction has a highly significant linear correlation with the amount of tissue ablated in the central region of the cornea. CONCLUSIONS: Pneumotonometric IOP readings after LASIK are reduced, without recovering preoperative values even 1 year after surgery, because of flap cutting and tissue removal in the central region of the cornea. The contribution of flap cutting is estimated to be (1.6+/-0.8) mm Hg, whereas ablation contributes an additional (0.029+/-0.003) mm Hg/microm of removed tissue. This effect should be considered when evaluating the accuracy of IOP measurements in LASIK patients who are at risk for developing glaucoma.