Tessa De Bie

Introduction
A suboptimal diet has been established as the single greatest risk factor for non-communicable diseases and is responsible for 1 in 5 premature deaths worldwide. To improve dietary quality, more emphasis could be placed on enhancing the nutritional quality of fruits and vegetables during crop selection, breeding and cultivation. While nutritional quality can be defined in terms of nutrient composition, there are also many, often unexplored, components present in our diet that might have beneficial health benefits beyond their nutritional value. Food composition databases are currently lacking information on such potentially bioactive food components and robust evidence regarding their health effects is often not available. Examples are flavonoids, anthocyanins, glucosinolates, indoles and catechins. This thesis introduces another plant component that has the potential to benefit human health: gamma-aminobutyric acid (GABA).

GABA is a non-proteinogenic amino acid and is best known as inhibitory neurotransmitter in the central nervous system. However, GABA is also present in food products. It is endogenously produced in plants like tomato, potato and melon and can also be produced during fermentation by microorganisms from its amino acid precursor glutamic acid. The GABA content can reach up to 10 grams per kilogram of product in for example matured cheeses. Interestingly, GABA receptors are not only present in the central nervous system but also in many organs and tissues throughout the body, suggesting physiologically relevant effects of peripherally present GABA. This was demonstrated in rodent studies that showed that GABA can have metabolic health effects in models for type 1 and type 2 diabetes.

Although it has been determined that GABA is taken up in the bloodstream after oral intake, it is still unclear how a food matrix affects its bioavailability and whether long-term GABA intake affects the circulating GABA concentration. Next to that, for the determination of the GABA concentration in human plasma, there is no standard, validated, analytical method available as is required for application in clinical trials. Finally, while GABA supplements are already marketed with health claims, the evidence regarding these health effects of oral GABA intake in humans is poor. This thesis therefore aimed to investigate the potential of GABA as a dietary plant component with health benefits.

Methods
Since tomato and potato have high GABA levels, while being consumed in high amounts across the world, a large collection of tomato and potato genotypes was screened for the GABA and glutamic acid levels in the fruits and tubers. Next to that, the effects of domestic cooking methods on the GABA and glutamic acid levels of tomato and potato preparations were assessed. To proceed, a UPLC-MS/MS method was developed for the simultaneous determination of GABA and glutamic acid in human plasma and the precision and accuracy of the method was validated. The method was applied to determine the relative bioavailability of GABA from a tomato matrix, and compared to GABA dissolved in water. In a randomized crossover trial, 11 healthy men were given either a liter of pureed tomato, an equivalent amount of GABA dissolved in water, glutamic acid dissolved in water, or plain water. Blood was drawn at 19 timepoints over 24 hours, to compare plasma kinetic parameters of GABA and glutamic acid. Ultimately, the health effects of 3 months GABA supplementation were explored in 52 individuals with prediabetes in a parallel, double-blind, randomized, placebo-controlled intervention study. As the primary outcome, the effects of GABA on the postprandial glucose response were investigated. As exploratory secondary outcomes, parameters of glycemic control (HbA1c, insulin, glucagon, mean amplitude of glycemic excursions, and standard deviation as measured with flash glucose monitoring), cardiovascular health (blood pressure, 24-hour blood pressure, circulating triglycerides, cholesterol) and sleep quality were measured.

Results
The screening showed that GABA content between different potato and tomato genotypes can vary substantially. Interestingly, the GABA content remains largely constant over different harvest years, suggesting a strong genetic determinant. Specifically, the potato cv. ‘Riviera’ and the tomato cv. ‘Madara’ have GABA contents of 0.7 and 1 gram/kg, respectively. In addition, the GABA content appears to be relatively tolerant to most common domestic cooking methods, and the bioavailability of GABA from pureed tomatoes is equivalent to GABA dissolved in water. After intake, the GABA plasma concentration peaked at, on average, 75 ng/mL after 30 minutes. In fasting condition, the GABA plasma concentration was found to be 16.7 ng/mL on average and stayed relatively stable (5.8 CV%) within individuals over the course of 4 weeks.

After 3 months of GABA supplementation, the postprandial glucose response was not affected by the intervention. However, exploratory secondary outcomes, like parameters of glycemic control and cardiovascular health, also showed no effect of GABA supplementation. However, a decrease in fasting plasma glucose and an improvement in sleep quality in individuals with prediabetes as compared to placebo was observed, although this was not significant after correction for the false discovery rate. The extent of the improvement in sleep quality seemed to be dependent on the individual’s sleep quality before the intervention.

Conclusion
We showed that specific tomato and potato genotypes are potentially substantial sources of GABA in view of their large contribution to diets across the world. Furthermore, GABA is taken up into the blood as efficiently from a tomato food matrix as from a solution in water. There are also some indications that after 3 months of supplementation, GABA might have some health benefits. GABA supplementation led to a modest reduction in fasting blood glucose and improved sleep quality in individuals with prediabetes although these were no longer significant findings after correction for the false discovery rate. Therefore, the evidence is currently not sufficient to recommend an increased dietary intake of GABA. The indications of beneficial effects that the research as presented in the current thesis provides, should be substantiated in more focused trials. Furthermore, more insight in the potential effects of GABA and their underlying mechanisms in peripheral tissues is necessary. Finally, different food products and a GABA-rich diet in general, should be used in further investigations, which should include different populations to translate the results to a real-world situation.