Diagnostics & Imaging Week Contributing Writer

NORWICH, UK – Recent efforts to optimize metabolomics technology is beginning to bear fruit in the discovery of novel biomarkers for disease. In general, the concentrations of metabolites are amplified relative to the proteins that prompted their formation, making them potentially more sensitive, both in diagnostics and as drug targets, than protein biomarkers.

"Metabolites are downstream from the protein, so you must have an amplification in concentration," Douglas Kell, director of the Manchester Centre for Integrative Systems Biology, said here at the British Association Science Festival last week. "Furthermore, you don't need to know the gene sequence, there are fewer metabolites than proteins, and they are generic, so once you can measure a metabolite, it is the same in every organism – which is not the case for proteins."

It is estimated that there are about 3,000 metabolites in the human metabolome. The issue is that there is a wide concentration range and it is very difficult to detect metabolites that occur at low concentrations.

Kell's group has developed an automated "robot scientist" that couples gas chromatography separation to mass spectrometry detection, and has optimized and refined the equipment to the point where it can discriminate 1,800 true metabolites.

Using the robot, blood samples from healthy and affected individuals have been analyzed to systematically uncover subsets of metabolites that are markers of particular diseases. For example, examining samples from women affected by pre-eclampsia in pregnancy and those who were not led to the discovery of three metabolites that distinguish sufferers.

"None of the [three metabolites] was visible [with our equipment] when we started; it is only as we improved the process that they were detected," Kell said.

Currently, pregnant women are sifted out as being at risk of pre-eclampsia by measuring blood pressure. "But this is more than a surrogate for blood pressure: You can look in the affected cohort only and follow the development of the disease," Kell said. More recently his group has found further metabolites linked to pre-eclampsia, making the biomarkers potentially even more sensitive.

Kell has discovered metabolites for Huntington's disease and for a number of cardiovascular diseases, also. "Many of these offer the possibility of novel interventions and of prognostic detection of diseases in their earliest stages, before they become life threatening," Kell said.

More precise detailing of live cells devised

NORWICH, UK – A team from Imperial College London and Cambridge University have devised a method for imaging cells without killing them, making it possible to watch biology in action. Among the processes recorded in real time are the congregating of protein complexes, microvilli responding to changes in osmotic pressure and viruses preparing to infect a cell.

The researches have used their equipment to deliver single molecules to precise locations on the surface of cells. They have "painted" nanoscale pictures by the molecule-by-molecule application of fluorescent DNA and simulated a heart attack in a petri dish by administering a single molecule of a toxin to a cardiomyocyte, prompting it to become arrhythmic.

"The big advantage of our technique is that it is non-contact," said David Klenerman, of Cambridge University, speaking at the BA Festival of Science here last week. "A lot of structural biology is based on examining dead cells. Now we can see live cells. It is not the highest resolution that has been achieved, but it is the highest achieved on a live cell."

The technique, called Scanning Ion Conductance Microscopy (SICM) uses a micropipette to deliver a small voltage to the surface of the cell. By scanning the pipette across the cell, it is possible to create an image of the surface. No special preparation is required and the cells can be imaged on standard culture dishes.

In addition, the pipette can be used to perform nanoscale measurements on the cell surface, such as recording a single ion channel, or for the controlled addition of reagents.

While SICM is not a new technique, the researchers have produced a micropipette that is smaller than any of its predecessors, enabling the resolution to go down to 10 nanometres.

Using fluorescent DNA, Klenerman and colleagues have painted the Cambridge University crest on a cell in lines that are under one micron in width. "This demonstrates very high control of where you put molecules and shows you can use the technique to do assays on individual living cells," Klenerman said.

The team has studied epithelial kidney cells, uncovering the same features that are seen in scanning electron micrographs, but then going on to follow the life history of microvilli, showing them responding to changes in osmotic pressure. Currently, the researchers are applying the technique to visualise ion channels in neurons, and Klenerman said this is leading to the development of a smart patch clamp technique, where the characteristic on/off behavior of ion channels can be followed live.

The micropipettes can be used also to apply pressure to a cell, pushing it down or sucking it up, without actually making contact. That means that it is possible to probe the mechanical properties of individual cells. Micropipettes can be constructed with a double barrel, to deliver two different molecules at the same time, and it also is possible to construct them with four and eight barrels.

The technique opens up the possibility of performing cell repairs, but Klenerman said that is some way off.

Vascular ultrasound screening effective

John Postley, MD, of the New York Physicians Group presented study results at the annual meeting of the World Congress of Cardiology (WCC; Antipolis, France) in Barcelona this week, demonstrating that vascular ultrasound screening can be a more accurate means of risk stratification for heart attack, particularly among women, than the Framingham Risk Score, the traditional method of identifying cardiovascular risk.

Postley used hand-carried ultrasound systems made by SonoSite (Bothell, Washington) in his study, titled "Gender Differences in the Relationship of Ultrasound Identified Atherosclerosis to Framingham Risk Score." A total of 120 patients were examined with ultrasound for the presence of carotid and femoral arterial plaque and the measurement of carotid intimal medial thickness (CIMT).

Among the 50 women in the study, Postley found no correlation between Framingham Risk Scoring (FRS) and the thickening of the endothelial lining of the carotid artery, or CIMT, a surrogate for cardiovascular disease events.

Additionally, while 72% of these women were at low risk for heart attack according to FRS, 50% of this group had incidence of plaque. Even in the absence of other risk factors, presence of plaque is a proven precursor to heart attack and stroke.

All CIMT measurements were made with a SonoSite TITAN system and SonoCalc IMT automated edge detection software.

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