Biomarkers help to understand disorders of the cell's energy generators
by Julie Boiko
Two billion years ago, a hungry proto-eukaryote - a cell lacking defined organelles that would allow it to make its own energy - engulfed an aerobic bacterium that uses oxygen to generate its own energy. Instead of digesting it, the proto-eukaryote formed a relationship with its helpful snack, feeding off the energy the bacterium produced by metabolizing its undigested food particles. This theory explains that with time, natural selection morphed the bacterium into what has now become an essential cellular organelle called the mitochondrion, the energy-producer of most every eukaryotic cell.
Because of their autonomous origins, mitochondria still maintain their own genomes, known as mitochondrial DNA (mtDNA), that are inherited maternally. Mitochondrial information, thus, has been used in population genetics and forensics testing, since it can be traced directly from offspring to mother. For the past eight years, Dr. Gregory Enns, Associate Professor of Pediatrics and Director of Stanford's Biochemical Genetics Program, and his colleagues have studied these exciting organelles and the diseases that result when they malfunction. Working directly with young patients affected by genetic mitochondrial diseases, Enns and his colleagues seek to understand the hallmarks of mitochondrial disorders and develop novel methods of diagnosis and treatment.
Mitochondrial disorders Enns' research team focuses on young patients with genetic metabolic disorders caused by mutations in both mtDNA and nuclear DNA. The mitochondria's inability to produce sufficient energy can primarily affect the function of neural and muscle tissue as well as other tissues of the body. The severity of mitochondrial disease is thought to be closely tied to the percentage of significantly mutated mitochondria present during embryonic development that was duplicated into the body's mature tissues. In other words, if there was a significant number of mutated mitochondria relative to normal mitochondria during early development, than the mature tissues would have a significant proportion of mutated mitochondria, and the disease would be significant. Also, if the mutated mitochondria were only present in specific cell lineages at the embryonic stage, then only specific regions of the body would develop to contain these mutated mitochondria.
Many mitochondrial and metabolic disorders worsen during times of intercurrent illness. For example, if a person with mitochondrial disease contracts the flu, then that illness can increase the severity of the disease. In this case, it could lead to catabolism - metabolic breakdown of healthy tissues - which could result in a vicious downward spiral in which mitochondria race to produce energy - unsuccessfully - and instead destroy themselves in the process. Enns likens this problem to Òstepping on the gas pedal with a flooded engineÓ. This mitochondrial breakdown often results in the failure of multiple organ systems. Enns' goal is to develop better ways to diagnose mitochondrial diseases and to monitor their progression.
Biomarkers
Because mutant mitochondria are often present in multiple tissue types, Enns points out that diagnosis of mitochondrial diseases can be challenging, as these disorders present themselves through a variety of symptoms. ÒRight now it is often difficult to diagnose, let alone treat, these conditions,Ó says Enns. ÒAlthough there are not many available therapies, we want to figure out if any treatments we're using actually work. To do that we need to have biomarkers of the disease itself.Ó
Metabolic biomarkers are clinically relevant disease features determined by measuring the levels of organic molecules in the body. At Stanford, Enns has sought to define such features for mitochondrial and other metabolic disorders to measure disease progression. In the clinic, Enns and his colleagues measure each patient's metabolites. These metabolites may or may not be already suspect in relation to a particular disease. Once sufficient data collection is achieved for a particular metabolic disease, researchers look for trends in metabolite levels correlating with disease presence and progression.
One example of a metabolic biomarker is glutathione. Glutathione works as an antioxidant, defending cells against the potentially destructive process of oxidative stress caused both by normal metabolism and environmental contaminants. When abnormal mitochondria don't efficiently produce energy, unused oxygen accumulates, producing reactive oxygen species (ROS) that produce oxidative damage by removing electrons from neighboring cellular molecules. Cells defend themselves by using glutathione to protect against damage from ROS. Enns utilizes glutathione levels as a biomarker in mitochondrial disease patients, determining the degree of oxidative stress and levels of glutathione in patients when they feel and appear healthy, and comparing them to the levels when the patients are acutely ill.
Since 2005, newborns in California have been screened for nearly 40 metabolic and other diseases through simultaneous measurement of various metabolites using tandem mass spectrometry, though mitochondrial disorders are currently not part of this expanded newborn screen. ÒIt's [this] technology that allows us to look at a lot of different metabolites at the same time...and makes it possible for us to screen kids for a lot of different conditions,Ó comments Enns on spectrometry's utility in catching diseases before patients become symptomatic. Although such technology has brought a flux of young patients with a variety of metabolic disorders to Stanford recently, Enns remarks that it is Ògratifying to see these kids coming in [to the clinic] instead of coming into the emergency room when they're in a coma.Ó
By defining biomarkers for mitochondrial diseases, Enns hopes that more metabolic diseases will be caught earlier and treated immediately rather than allowed to silently persist until it is too late for medical intervention. Additionally, further identification of biomarkers will allow physicians to monitor the progression of mitochondrial disorders more easily and make appropriate treatment decisions.
Promising research
Mitochondrial disease research has a long way to go, both in its understanding of mitochondrial genetics and in its clinical applications of defining biomarkers. Work published by Enns and other researchers (Molecular Genetics and Metabolism, 2003) indicates that mtDNA mutations may cause diseases long believed to be largely unrelated to impaired mitochondrial function, inclusding diabetes, hypothyroidism, and possibly even some cancers. It is thought that dysfunctional mitochondria may produce toxic reactive oxygen and nitrogen species that set off a destructive chain reaction leading to such disorders. Enns hopes that his work with pediatric mitochondrial disease patients will serve as a paradigm by which clinical researchers can develop treatments and novel methods for diagnosis. ÒWe're catching some [patients] before they have a metabolic crisis,Ó says Enns. He looks forward to the day when knowledge of mitochondrial malfunctions will allow physicians to diagnose and treat patients with mitochondrial disease even before they begin expressing symptoms.