A new discovery at the University of Louisville may help scientists test the effects of exposure to green plants “greenness” on health. Pawel Lorkiewicz, associate professor of chemistry and environmental medicine at , measured a person’s exposure to trees and plants by testing their urine.

Limonene is a nontoxic substance emitted into the air by many trees and plants. Concentrated limonene has a lemony smell and is used in perfumes, foods, cleaning and other household products. When a person breathes concentrated limonene, the body breaks it down into metabolites which can be measured in their urine.

These metabolites also are in a person’s urine after they spend time in a forest, . This means the metabolites can be used to measure a person’s exposure to trees and plants. Comparing this exposure with health information may help researchers more accurately determine whether time spent in nature is beneficial for an individual’s health.

“Even though we may not smell limonene exactly, usually we can tell that we are surrounded by trees and greenness because of the smell. Parks and forests, or even meadows or larger green spaces have a characteristic smell because of plant volatile organic compounds such as limonene,” Lorkiewicz said.

For several years, Envirome Institute researchers have been studying the health effects of living near trees and greenness. As part of the , they planted trees and shrubs in specific neighborhoods, measuring changes in the residents’ health compared with those living in areas with fewer trees. But the researchers found it challenging to accurately measure an individual’s actual exposure to trees. There may be many trees where they live, but they spend much of their time working in tree-sparse locations or indoors, insulated from trees and plants.

So the team searched for a way to measure how much time an individual spent around greenness. Because many trees and plants release significant amounts of limonene into the air, they developed a method to test for limonene exposure using urine, which is easy to collect.

After testing the urine of people who smelled pure limonene from a vial, they compared those results with urine from people who spent time walking in a forest. They found the same metabolites in the urine of both groups, tagging three of them as reliable biomarkers of exposure to greenness.

“It was very exciting. That’s what constitutes a biomarker, something was released by trees in real life, metabolized by our bodies and found in our urine, not just the result of smelling a pure chemical in a lab,” Lorkiewicz said. “So, we objectively can tell when someone is not working in an office at a desk, but they actually go outside and are surrounded by greenness.”

Keith said the biomarker for greenness exposure is a breakthrough for the Green Heart Project, the Trager MicroForest project in downtown Louisville and other studies.

“We have been working with the overall premise that trees affect health in a positive way. Our mission in the Green Heart Project is to understand in some ways how they do that,” she said. “We have turned to the idea of using the human as the biomonitor of greenness in the human. We hope to use this to see changes in people’s overall exposure to greenness as we plant trees and determine whether those changes correlate to changes in health.”

To assess people’s exposure to trees and plants, Keith combines the limonene biomarker test with satellite images showing tree density where people live and surveys of their time spent outdoors and compares the exposure information with health data such as blood pressure and heart rate.

In additional research, Keith and her team are using the biomarker to compare the health effects of limonene and other compounds emitted by plants on the body.

The $500,000 gift to the Division of Cardiovascular Medicine at the UofL School of Medicine establishes the James Ryan Fund for Cardiovascular Biomarker Research. Ryan pledged the gift before he passed away at age 81 in 2018.

“Thanks to Jim, we can help physicians everywhere in diagnosing what is one of the most common causes of death in the world,” DeFilippis said. “He was always interested in others and what their dreams were, their goals in life, their happiness and how he could help.”

The fund will support annual operating expenses for the research led by DeFilippis, who is director of Cardiovascular Disease Prevention and an associate professor in cardiovascular medicine at UofL. DeFilippis is an expert in cardiovascular diseases and cardiac intensive care. His research focuses on cardiovascular risk prediction and the identification of biomarkers that will allow physicians to diagnose the cause of different types of heart attacks, also called myocardial infarction.

A heart attack is death of heart muscle. There are many causes, including the most well-recognized that occurs when one of the heart’s coronary arteries is suddenly blocked or has very slow blood flow secondary to the formation of a blood clot.

Atherosclerotic cardiovascular disease progresses over time, allowing time for screening and early detection. Advances in biomarker research and other developments have led to more sensitive screening methods and a greater emphasis on early detection and diagnosis.

“We’re working to develop biomarkers that will allow clinicians to differentiate among the many different types of heart attacks that can occur,” DeFilippis said. “Not all are the same, and even in the medical community that’s not always given much thought. But if we can differentiate, it will help us better treat patients, allowing us to limit or stop heart damage.”

A biomarker is a biological characteristic that can be measured and evaluated as an indicator of normal biological or pathological processes or a response to a therapeutic intervention. Examples include patterns of gene expression, levels of a particular protein in body fluids or changes in electrical activity in the heart.

Ryan, an active philanthropist and retired insurance executive, suffered from a heart condition himself. A native of Pittsburgh who graduated from UofL and lived most of his life in Louisville, he was on a quest to ensure his estate went to good use. He and DeFilippis forged a friendship through their activities with the American Heart Association. He visited DeFilippis’ lab within the Division of Cardiovascular Medicine several times, even bringing a group of friends.

“He was really involved – he was more than a donor,” DeFilippis said. “He gave advice and offered assistance in business, enabling us to partner with industry to bring our discoveries to market. We really enjoyed his company. The two of us went to lunch several times and talked about lots of things – the lab, his struggles with congestive heart failure and his bucket list. He had a great sense of humor.

“He was an extraordinarily happy man with a deep voice that was very distinctive,” DeFilippis said. “We will certainly miss his presence and his inspiring words about the work we were doing. We take it as a tremendous compliment that he chose to donate to us. Jim was looking for a quality program, great ideas and things that really make sense to improve the health of world.

DeFilippis and one of his colleagues, Patrick Trainor, PhD, recently were invited editorialists for a new study published in the Annals of Internal Medicine that indicates current risk calculators for heart attack and stroke can sometimes be wrong, significantly overestimating some people’s risk while underestimating others’ risk. As a result, many people may be unnecessarily taking medication to control risk factors such as high cholesterol and high blood pressure, and others may not be getting enough treatment. The researchers developed a new risk calculation method with a more sophisticated statistical model and newer population data, suggesting it could be more accurate.