Further studies follow. The full research write up is below:
Organic Acids Supplementation to Augment Mitochondrial Performance
Accessing Mitochondrial Efficiency in Cultured Mammalian Cells
Shuk Yee Ngan, Tanya Burkhart and Frank Merante Seneca College of Applied Arts and Technology
November 2016
Introduction
Serum lactic acid levels as a means of monitoring the degree of substrate flux through the Kreb cycle (Citric Acid Cycle) and hence achieve indirect measure of mitochondrial performance is a field of interest in exercise science and physiological studies. Progressive exertion and exercise can increase blood lactate levels associated with change in ventilation and metabolic acidosis1, which eventually affect exercise performance. Lactic acid fermentation is a result of metabolic alterations within the cell.
In glycolysis, one glucose molecule is broken down to yield two molecules of pyruvate as a means of generating reduced NADH and ultimately cellular ATP. Subsequently, the pyruvate and NADH are used to regenerate NAD+ by aerobic respiration in mitochondria or lactic acid fermentation in cytosol. Although lactate fermentation is commonly known as an anaerobic reaction, lactate fermentation is not only driven by inadequate oxygen supply, but during exertion, glycolysis produce excessive pyruvate concomitantly increasing the cytosolic NADH/NAD+ ratio and shifting the cellular equilibrium to lactic acid production2. Cytosolic lactate is released in blood and regulated by buffering system in blood and fibers. When lactate level exceed the buffering capacity, muscle function and exercise performances are affected. Therefore, the delay of lactate accumulation may act to prolong endurance events during periods of necessity. In addition to lactate levels, the lactate/pyruvate ratio directly reflects the NAD+/NADH ratio, which in turn is an indicator of the mitochondrial performance.
In this study, in vitro moderate throughput enzymatic methods were developed and optimized to enable the assessment of extracellular lactate levels produced during various metabolic interventions. Enzymatic assessment of lactic acid suggests that Kreb cycle intermediates (pyruvate, citrate, malate and succinate) and ascorbic acid can reduce lactate production and promote mitochondrial performance even in the presence of fermentable carbon sources, namely glucose.
Methods
BHK Cell Culture Preparation
Baby Hamster Kidney (BHK) cells were cultured in 6-well cell culture plates with MEM solution supplemented 10% bovine calf serum and 2mM glutamine. Seeded plates were incubated in incubator with 5% CO2 at 37°C until confluent. MEM was removed from confluent cells. The cells were rinsed twice with Phosphate Buffered Saline (PBS), and incubated with PBS for one hour to deplete storage nutrients. Following the one hour incubation period, 1mL of test substrates, prepared in PBS were added and incubated for an additional one hour. The extracellular solution was collected and immediately frozen and the cells were harvested with cell scraper.
Lactate Enzymatic Assay
50uL of cell supernatant were tested with approximately 7 units of lactate dehydrogenase in Tris buffer (2.0 M, pH 9.2) supplemented with β-NAD+. A SpectraMax 190 microplate reader was used to measure absorbance at 340 nm. Lactate production rate is calculated by dividing lactate concentration by reaction time and protein concentration.
Bradford Assay for Protein Determination
A bovine serum albumin standard curve was generated to assess protein concentration in the various samples. Essentially, 5uL of each BSA standard was added to 250uL of Bradford reagent in 96 well plate. Absorbance was determined at 595nm by 96-well plate reader.
Results
Organic Acids Reduce Cellular Lactate Production
The potential for metabolic intervention resulting from the inclusion of various small molecule organic acids was investigated. Test conditions relative to glucose, a commonly used additive to sports recovery and support drinks. Control interventions using glucose exhibited lactate production levels of 2.348nmol/min/μg, while the inclusion of various organic acids tended to decrease lactate production, and in some cases significantly so (Figure 1). For example, succinate added to the BHK cultures in the presence of glucose, resulted in a statistically significant reduction in cellular lactate production (p >0.05) for 17%.
Figure 1. Average lactate acid produced in 60mins in different treatment conditions. Asterisk (*) columns represents statistically significant reduction in relative to glucose.
Ascorbic Acid Mediates Lactate Production
Most notable, ascorbic acid also reduces lactate production in the presence of Kreb cycle intermediates. For example, ascorbic acid, in the presence of Kreb cycle intermediates (pyruvate, malate, citrate, and succinate) result in significant reduction in cellular lactate production (p>0.05) comparing to glucose for 21%. Additionally, the presence of ascorbic acid in Kreb cycle intermediates (pyruvate, malate, citrate) reduces cellular lactate production by 22%, when compared to the absence of ascorbic acid (p>0.05) (Figure 2).
Figure 2. Average lactate acid produced in 60mins in treatment conditions of Kreb cycle intermediate with (dark blue) or without (light blue) ascorbic acid. Asterisk (*) columns represents statistically significant reduction in relative to condition without ascorbic acid.
Discussion
Based on the results shown in Figure 1, Kreb Cycle intermediates can modulate lactate production in BHK cells, suggesting that metabolic intervention using Citric Acid cycle intermediates is feasible and achievable. Although the exact mechanism of lactate reduction is unclear, supplementation with Kreb cycle intermediates may augment the optimization of substrate flow through the mitochondria. Furthermore, such intervention may improve the efficiency of oxidative metabolism, facilitating improved mitochondrial performance and utilization organic acid as mitochondrial fuels. Collectively, the cytosolic pyruvate and NADH level can be maintained at an optimal state to avoid lactate accumulation and hence maintain efficient ATP production by oxidative means.
Although ascorbic acid is not generally considered a Kreb cycle intermediate, but rather simply an antioxidant reagent, the data gathered herein suggests that ascorbic acid can promote electron transport in mitochondrial matrix and act to improve mitochondrial performance.
A limitation of this study is that immortalized BHK cell cultures may not precisely model exercised cells, particularly in their metabolic demand, but are nevertheless a good model for the aerobic needs akin to human kidney cells. Most notably is that kidney cells are not as metabolically active as muscle, therefore a future study will proceed with a muscle cell line which exhibits an increased metabolic demand.