The tears of people with major depressive disorder, or clinical depression, have a different chemical composition than the tears of nondepressed people, according to a recent study that highlighted tear fluid's potential for the simple, rapid diagnosis of mood disorders.

Tear fluid is considered a source of biomarkers, or biological markers, which are seen as indicators of normal biological processes that can be objectively measured, similar to blood pressure or heart rate. In a paper published March 29 in the Journal of Psychiatric Research, a group of researchers from Slovakia explored how the analysis of tear fluid may facilitate the diagnosis of mood disorders, such as depression or bipolar disorder, and has potential for future integration in bioinformatic pipelines and personalized medicine.

More than 264 million people of all ages suffer from depression around the world, and between 76% and 85% of people in low- and middle-income countries do not receive treatment for their depression. They face barriers such as a lack of resources and a lack of trained health care professionals, and in all countries, depression is often misidentified or treated incorrectly because its diagnosis relies on subjective conversations between doctor and patient. 

Kristína Krajčíková, the first author of the paper, who completed the research as part of her Ph.D. at Pavol Jozef Šafárik University, told The Academic Times that she built the study on previous research of biomarkers in tear fluid, but that these issues had not previously been studied in the context of major depressive disorder. She and her colleagues collected tear fluid from a sample of depressed adults and compared it with tear fluid from a sample of healthy controls and found that there were discernible differences between the two groups.

"The most important finding was that [the] tear fluid proteome is really significantly changed in depressive subjects," Krajčíková said. The proteome is the totality of proteins expressed by a cell, tissue or organism at particular times and under particular conditions. "It forms the base for future studies, demonstrating that tear-fluid biomarker studies are worth the time and effort," Krajčíková continued.

The researchers collected basal tear fluid from a sample of 10 depressed adults and 21 healthy adults. Basal tears are the basic functional tears that lubricate and protect the cornea, which covers the front portion of the eye and refracts light. Krajčíková said that basal tears are important because the human body produces them constantly in a low amount, but collecting them is challenging because of the small volume. Other types of tears, such as emotional tears, are highly variable in composition, making them generally unsuitable for biomarker research. 

The team collected basal tears in two ways: by flushing with a sterile saline solution and by applying cellular microsponges to the surface of the eye without touching either the cornea or the conjunctiva, the thin membrane that covers the front surface of the eye and inner surface of the eyelids.

After collecting the tear fluid, the researchers used both standard techniques and specialized spectroscopic methods to analyze their composition. Spectroscopic methods have not been used in psychiatric research before, Krajčíková said, but they have proved capable of detecting subtle neurobiological abnormalities in major depressive disorder.

"In this study, we focused on the identification of potential biomarker candidates in subjects with [major depressive disorder] from human tear fluid," the authors reported in the paper. "Because tear fluid is more accessible (non-invasive sampling) and less complex than other body fluids (e.g. plasma or serum), it shows great potential as a sampling material."

The more standard methods they used included matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is an ionization technique involving a laser energy-absorbing matrix that generates singly charged ions from large molecules. More unusually, they used more obscure techniques, such as synchronous fluorescence spectroscopy, a spectral method, as well as zymography, a technique used to detect hydrolytic enzymes, according to the paper.  

The authors explained that they chose to use those spectral methods in this study because of their rising popularity in human body-fluid research over the past two decades. "Their complex approach enables identification of potential biomarkers for particular psychiatric disorders," they said. "Many of these methods have already been helpful in better understanding of psychiatric disorders from the molecular point of view."

Tear fluid contains water, salts, proteins, lipids, hormones, vitamins, nucleic acids and other diagnostically important molecules, according to the paper. The researchers specifically looked to identify potential protein biomarkers in the tear fluid of people with depression, which is why they assessed changes in the tear-fluid proteome. Their analyses revealed significant changes of proteins as a complex in the tear fluid of the depressed participants 

In particular, the researchers identified key differences in the expression of a protein known as matrix metalloproteinase 9 (MMP-9). Different forms of MMP-9 occurred only in the tear fluid of the depressed participants, and no forms of MMP-9 were detected in healthy controls.

"Matrix metalloproteinases are of special interest in [major depressive disorder], because on one hand, they regulate neurogenesis, oligodendrogenesis and brain plasticity in [the] developing brain, and on the other hand, they regulate pathological processes like neurodegeneration, vascular and inflammatory disorders," the authors reported in the paper.

The authors noted that the spectroscopic methods employed in their study are experimental and not yet commonly used in clinical diagnostic laboratories, but their findings signal such techniques' potential for the fast and minimally invasive diagnosis of major depressive disorder using tears. 

The current state-of-the-art approach for psychiatric diagnostics is a standardized psychiatric interview, involving questionnaires and self-assessment scales. But as the authors point out, this method can be subjective. They recommend that a combination of their proposed spectroscopic methods "may be helpful in providing a more objective and valid psychiatric assessment" in clinical settings. "Our preliminary results show that human tear fluid has great potential as a sampling material in clinical diagnostics, including psychiatric research," the authors added.

The authors also noted the limitations of this pathbreaking work: "Because of the relatively small sample size, follow-up studies and further investigations need to be performed to confirm the results and facilitate their evaluation and validation."

Krajčíková called her study a first step toward integrating tear fluid into the diagnosis of mood disorders. She suggested that future research should explore ideas, such as the development of a software to allow diagnostics of major depressive disorder from tear fluid using the spectroscopic methods, and answer critical remaining questions. For example, which protein biomarker for major depressive disorder occurs in tear fluid, and what does it reveal about the mechanism of the disease?

"With the massive development of bioinformatics, we believe it is not necessary to look for a specific biomarker of major depressive disorder but software that would consider the outcomes from all spectroscopic methods," Krajčíková explained. "This option allows [for] enhancing the specificity and sensitivity of the diagnostic test. And maybe it would be a way towards personalized medicine in this field."

The study, "Tear fluid biomarkers in major depressive disorder: Potential of spectral methods in biomarker discovery," published March 29 in the Journal of Psychiatric Research, was authored by Kristína Krajčíková, Daria Kondrakhova, Jana Mašlanková, Marek Stupák, Ivan Talian, Tatiana Kimáková, Vladimír Komanický, Katarína Dubayová and Vladimíra Tomečková, Pavol Jozef Šafárik University; Erika Semančíková and Eva Pálová, L. Pasteur University Hospital and EPAMED s.r.o; Katarína Zakutanská and Natália Tomašovičová, Slovak Academy of Sciences; and Dagmar Breznoščáková and Juraj Semančík, L. Pasteur University Hospital.