Researchers identify lipid profile characteristic of newly diagnosed type 1 diabetes

The Editors-in-Chief of Clinical Biochemistry and Clinica Chimica Acta selected the paper for the distinction and included it in a booklet distributed at international meetings sponsored by the International Federation of Clinical Chemistry.

T1DM affects more than 1 million individuals in the United States alone. Currently, the best approach for predicting those at risk for developing T1DM before symptoms appear is by measuring autoantibodies to islet cell antigens in the pancreas.
Lipids 101
Lipids are one of the four major molecular components of biological organisms, along with proteins, sugars, and nucleic acids. They store energy, make up the structures of cells, and participate in cell signaling.  The "lipidome" refers to all lipids in cells and "lipidomics" to the measurement thereof. Autoantibodies cause many autoimmune diseases. Multiple autoantibody positives and their persistence are unequivocally related to the risk of progression to T1DM. However, autoantibodies are difficult to measure with consistent sensitivity and specificity, and assay performance varies considerably among laboratories. Sound methods are needed to identify new biomarkers that predict T1DM development and can also be transferred to other laboratories.

To improve and standardize measurement of autoantibodies associated with T1DM and identify novel protein biomarkers, the Centers for Disease Control and Prevention (CDC) and the Immunology of Diabetes Society created the Diabetes Antibody Standardization Program (DASP). Scientists at PNNL and the CDC previously conducted a global proteomics analysis of plasma and serum samples from the DASP and identified five candidates in a sample subset (Metz et al. 2008).

The role of ZAG: Scientists found that one protein in particular, zinc alpha-2-glycoprotein (ZAG), was strongly upregulated, or increased, in individuals with T1DM. ZAG, a member of the immunoglobulin superfamily, is responsible for lipid mobilization. This means that increased levels of ZAG in patients may indicate a system-wide mobilization of lipids for energy production, particularly because these individuals lack endogenous insulin and cannot rely on blood glucose (sugar) for their energy needs. The scientists hypothesized that perturbations, or changes, may be present in the blood lipidome of individuals with newly diagnosed T1DM.

To test their hypothesis, they performed lipidomics analyses on the same DASP samples to identify perturbations in the lipids of individuals with recently diagnosed T1DM and to potentially identify a lipid profile that could predict or diagnose the disease.

"When discussing lipids in the context of personal health, most people think of total cholesterol, HDL, LDL, and triglycerides, which are the lipids typically measured in a blood lipid panel at clinics and hospitals," said PNNL chemist Dr. Thomas Metz. "They don't realize that there is a broad diversity of lipids in blood and tissues—thousands of individual lipid molecular species comprising about a dozen or so major classes.

"For example, HDL and LDL themselves are comprised of hundreds of molecular species of cholesterol esters and triglycerides. So, while the typical clinic blood lipid panel may not indicate differences in total cholesterol, HDL, LDL, and triglycerides, there may be dramatic changes at the level of lipid molecular species. These are the changes that only mass spectrometry can identify with sufficient sensitivity and throughput."

The scientists used capillary liquid chromatography (LC) coupled with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) and the accurate mass and time (AMT) tag approach developed at PNNL to identify and quantify lipids present in healthy and diabetic individuals. The AMT tag approach relies on initial, low-throughput shotgun LC-MS/MS analyses to populate a database of identified molecules followed by higher throughput and more quantitative LC-MS analyses.

They identified more than 559 lipids that were significantly different (q < 0.05) between control and patient samples, of which 55 were structurally identified by matching to entries in a blood plasma lipid AMT tag database. These lipids may represent a profile characteristic of newly diagnosed T1DM.

"The patients in our study had well-controlled blood glucose levels, so their blood lipid panels showed no differences in total cholesterol, HDL, LDL, and triglycerides compared to the controls," Metz said. "In that respect, it is very exciting that we identified so many lipid molecular species that showed statistically significant differences between the patients and controls. It really highlights an area worth further study in the context of T1DM."

More information: Sorensen CM, J Ding, Q Zhang, T Alquier, R Zhao, PW Mueller, RD Smith, and TO Metz. 2010. "Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: lipidomics analysis of a Diabetes Antibody Standardization Program sample subset." Clinical Biochemistry 43(12):948-956.

Metz TO, W-J Qian, JM Jacobs, MA Gritsenko, RJ Moore, AD Polpitiya, ME Monroe, DG Camp II, PW Mueller, and RD Smith. 2008. "Application of Proteomics in the Discovery of Candidate Protein Biomarkers in a Diabetes Autoantibody Standardization Program Sample Subset." Journal of Proteome Research 7(2):698-707.

Provided by Pacific Northwest National Laboratory (news : web)

Revealing the inner workings of cells with newly developed Raman microscopy technique

Living cells are virtuosos of chemistry. At any one time, countless chemical reactions are taking place within each cell. For researchers trying to understand how cells function, unraveling this complex chemistry is an ongoing challenge. The process, however, could soon become a little more straightforward. Mikiko Sodeoka and colleagues at the RIKEN Advanced Science Institute at Wako, in collaboration with a team led by Katsumasa Fujita and Satoshi Kawata at Osaka University, have demonstrated how to tag molecules in a way that promises to be much more versatile than current methods.

Traditionally, researchers looking to study the role of a particular small molecule within a cell have tagged it with a fluorescent marker. Using a fluorescence microscope, the tagged substance can be followed as it moves around the cell. However, fluorescent tags are bulky, and so can disrupt the molecule’s normal cellular interactions. To get around this problem, molecules can sometimes be tagged after reaching their destination within the cell, but this technique only works in a limited number of cases.

Sodeoka and her team have now shown that a simple chemical substituent called an alkyne, which consists of just two carbon atoms joined together by a triple bond, can replace the bulky fluorescent tag. Their imaging technique relies on the fact that alkynes scatter a particular wavelength of light when irradiated with a laser—a process known as Raman scattering—which can be detected using a Raman microscope. No other cellular components scatter light at this wavelength, giving a clear picture of the molecule within the cell.

To demonstrate the potential of their technique, the researchers used an alkyne-tagged component of DNA known as EdU. They then used a Raman microscope developed by Fujita and his team to follow a group of replicating cells as they incorporated EdU into their DNA (Fig. 1). The technique took some work to optimize, says Sodeoka. “We were very happy when we could finally see the time-series pictures of the incorporation of EdU into DNA.”

The EdU experiment is just a proof of principle, Sodeoka adds. “At this point, the sensitivity of the alkyne tag using Raman microscopy is lower than fluorescent imaging,” she says. To improve the sensitivity, the team is working to optimize the attachment of the alkyne tag, and also to improve the Raman microscope itself. “If the sensitivity problem is solved, Raman imaging using alkynes as a small tag could become a powerful tool,” she concludes.

More information: Yamakoshi, H., et al. Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy. Journal of the American Chemical Society 133, 6102–6105 (2011).

Provided by RIKEN (news : web)

Newly identified spider toxin may help uncover novel ways of treating pain and human diseases

Spider venom toxins are useful tools for exploring how ion channels operate in the body. These channels control the flow of ions across cell membranes, and are key components in a wide variety of biological processes and human diseases.

A newly identified toxin from the American Funnel Web spider acts on T-type and N-type calcium channels, researchers from the University of California at Riverside have discovered. The toxin offers a new target for studying T-type channels, which play a role in congestive heart failure, hypertension, epilepsy and pain.

"The blocking mechanism of the toxin is different from classical pore blocker toxins and voltage modifier toxins," says lead researcher Xiao Zhang, a postdoc at the Del Webb Center for Neuroscience in La Jolla, Calif. "It indicates a new toxin blocking mechanism on voltage-gated ion channels."

Zhang purified the toxin and created a recombinant version as part of his doctoral research at the University of California, Riverside. "If we can develop a calcium-channel blocker based on this toxin, we could have a new way to identify how these channels work and develop drugs for treating pain and disease," says Zhang.

More information: The presentation, "A Spider Toxin and its Recombinant Isoform Block T-type and N-type Calcium Channels with High Affinity" by Xiao Zhang, Li Dai, and Michael E. Adams is at 1:00 p.m. on Wednesday, March 9, 2011 in the Baltimore Convention Center, Room 307. ABSTRACT: http://tinyurl.com/4nhp7m6

Provided by American Institute of Physics