“In genomics and proteomics, data is quite species specific,” says Hans-Peter Deigner, Ph.D., director of biomarker research at Biocrates, “but in metabolomics investigations, studies on animal models translate well to humans.”
According to Dr. Deigner, metabolomics measures metabolite differences in biological fluids and tissues, providing the closest link to the various phenotypic responses. The goal is to span the gap between genotype and phenotype through an analysis that joins the individual biochemical reaction to the critical factors of drugs, nutrition, and environment. Since metabolites are the quantifiable molecules with the closest link to phenotype, toxic responses to a drug or disease prevalence are predicted by differences in the concentrations of the relevant metabolites.
The Biocrates team is investigating approximately 20 lipid analytes that vary in response to bleeding and occlusion brought on by stroke. “In addition, we follow the effects of aging in our mouse model, since the reaction to stroke changes as a function of age.” Using the mouse model, the investigators are able to sample brain tissue as well as serum from affected animals, and correlate these observations with data from patients.
Dr. Deigner and his colleagues are well aware of the limitations of omics studies. “We have learned from other’s failures and have built a robust statistical framework and analytical process for biomarker validation,” he says. “Because the number of individuals needed for a meaningful clinical trail is critical, we are working closely with our partners to ensure that we have a large candidate base sufficient to properly validate our studies.”
Markers for AIDS
In the course of an AIDS infection, the HIV virus can infiltrate the central nervous system and bring about dementia and encephalitis. With antiretroviral therapy, patients are living longer and may encounter significant neurocognitive disorders. According to William Wikoff, Ph.D., research associate at Scripps Research Institute, metabolomic profiling has the potential to provide early diagnosis of neural involvement and to predict the progression of the condition to a full-blown CNS disease. Moreover, it may eventually be possible to determine response to treatment and provide insights into the pathogenesis, treatment, and prevention of AIDS.
Although there have been numerous proteomic investigations of urine, serum, and CNS fluid from patients, these studies have provided few insights, driving the search for alternative screening tools. For neuroinfectious, neurodegenerative, and psychiatric disorders, the cellular and phenotypic complexity of the brain has hindered biomarker identification. Dr. Wikoff and his colleagues have invoked a new strategy, focusing on metabolomic studies to reveal, until now, undiscovered markers.
The Scripps group uses SIV infection of rhesus macaque monkeys as a model for human AIDS. The animals show the same pattern of symptoms, including dementia, CNS degeneration, and encephalitis observed in human AIDS patients. The experimental design that the team followed involved collecting cerebrospinal fluid before and after viral infection.
“The screening platform used a nontargeted, mass-based metabolomics approach with online data base screening to identify metabolites,” explains Dr. Wikoff.
Because the blood-brain barrier is partially compromised during HIV infection, the concentration of albumin and many metabolites such as fatty acids and phospholipids are increased. For instance, carnitine and acylcarnitines are elevated in infection as are various other unknown metabolites.
The Scripps researchers have searched for molecules that are specifically altered in monkeys showing encephalitis in the course of their SIV infection. They subsequently performed a microarray analysis to track down changes that relate to lipids or lipid processing, and identified increased expression of phospholipases in the brains of animals with encephalitis. Through the use of in situ hybridization and immunohistochemistry of brain tissue from infected animals it was determined that elevated phopholipases occurred in astrocytes and in the ventricles of the choroid plexus.
“We believe the identification of specific metabolites illustrates the potential of mass-based metabolomics to elucidate neurodegenerative diseases,” Dr. Wikoff concludes.
Various omics approaches have been used to develop databases of biomarkers for a number of years, yet it has proven extremely difficult to assemble consistent bodies of information that can be used to develop new disease biomarkers. In numerous cases, studies cannot be replicated and putative biomarkers cannot be validated. There are a number of reasons for these failures, but inadequate sample size, failure to control methodological variation, and inadequate quality assurance have been contributing factors.
Success within the omics field is absolutely essential for the development of a robust biomarkers. If we have learned anything from the advance of biomedical science in the last half century it is that diseases that are virtually untreatable by any available technology when they reach an advanced stage can be cured by essentially trivial and low-tech intervention during their early stages. It is virtually assured that no significant progress will be made until rapid, sensitive, cheap, and noninvasive methods are available for early detection.