STUDYING METALS IN THE BRAIN

By Mary Finnegan, Postgraduate researcher, School of Engineering

Could the presence of iron in brain cells help to explain why some people suffer from Alzheimer’s disease? Mary Finnegan, PhD student at the University of Warwick, is using specialist technology called the Diamond accelerator to improve our understanding of the relationship between Alzheimer's and trace metals, with the hope that diagnosis and treatment can be improved in the future.

Iron is important in cells, acting as a catalyst for many chemical reactions. It and copper, zinc, potassium, manganese, magnesium and many others are critical for our bodies to stay healthy. Most of these metals are at such low levels in the body - around one in a million, or even one in a billion atoms - that they are known as trace metals.

Although these metals are essential, they can be harmful if the levels become too high or they are in a dangerous chemical form. To be used properly by the body iron needs to switch between the more reactive Fe2+ and the less reactive Fe3+. However, too much reactive Fe2+ can be toxic to cells and so proteins are used to transport iron around the body and store it as the less reactive Fe3+.

If the way that metals are managed by the body is disrupted, this can cause disease. Researchers have found small changes in the quantity of iron in some of the regions of the brain affected by Alzheimer’s disease and also changes in the quantity of proteins that regulate iron. It is very difficult to measure changes in trace metals in the body as the quantities are so small. Traditional methods involve taking a block of tissue, dissolving it and using a spectrometer to measure the total concentration of the different metals in that block. However this does not provide any information about where the changes in the metals occur or what form the metal is taking. Is there more iron, copper, and/or zinc in the cells that are dying? Is there more reactive, and therefore more toxic, Fe2+ present in Alzheimer’s disease than in people with healthy brains?

This is where the Diamond Light Source can help. Diamond is the UK’s synchrotron and scientists can use the special X-rays produced at Diamond to detect metals in biological tissues.

Diamond Light Source

The ‘light’ produced by the Diamond accelerator is not just visible light. It covers much of the electromagnetic spectrum, from infra-red to X-rays.

A synchrotron is a particle accelerator designed to produce very bright light. There are four steps to producing the synchrotron light at Diamond:

1. Electrons are produced by an electron gun, and a linear accelerator (linac) uses high voltages to accelerate the electrons.

2. These electrons are fed into a small booster synchrotron and accelerated until they are travelling close to the speed of light.

3. The electrons are now passed into the storage ring of the main synchrotron. The storage ring is not actually a circle, but a pentagon made up of straight lines. At the junction of these straight sections are bending magnets which change the direction of the electrons and cause them to give off energy in the form of light.

4. This light can be channelled out of the storage ring, at points called beamlines, and used for experiments.

Different bending magnets mean that Diamond can produce light with a range of wavelengths from infra-red, to visible, to ultra-violet to X-rays. The synchrotron light is extremely bright – up to a 100 billion times brighter than the sun – and can be tuned to a very narrow range of wavelengths (energy) at each beamline, depending on what experiments are to be carried out.

To detect metals in biological tissues scientists can make use of a phenomenon called X-ray fluorescence.

To detect metals in biological tissues scientists can make use of a phenomenon called X-ray fluorescence. X-ray fluorescence occurs when a high energy X-ray ejects an inner electron (close to the nucleus) from an atom. This creates a ‘hole’ which is unstable and an outer electron falls into the hole to replace the ejected electron. This produces and emits a photon (another X-ray) with an energy specific to the particular element from which the emitted photon was produced.

Postmortem brain tissue, left to medical research by someone who had Alzheimer’s disease, is cut into thin sections of around 30 µm thick and X-ray fluorescence is used to look for metals in the tissue. The beam of X-rays is tuned to an energy of 10 000 eV and focused to a square spot size of between 60 and 3 µm. The X-ray beam scans across the sample and a detector collects the X-rays emitted at each point. The detector can detect X-rays across a wide energy range and so produces a spectrum of emitted energies. The spectrum will contain peaks where there is an element fluorescing at that energy. For example, iron fluoresces at an energy of 6403 eV so if there are X-rays detected at this energy we know iron is present. By looking at how the intensity (brightness) of the X-rays at 6403 eV changes across the sample, the variation in iron concentration can be observed.

Sample preparation

The way the samples are prepared is very important as this technique is very sensitive and any contamination on the sample could give false results or ‘shine’ much more brightly than trace metals in the tissue, masking their signal. The tissue must be cut in a very clean environment with a non-metal knife. The section is mounted onto a slide made of quartz – glass slides cannot be used for most of these measurements as glass often contains randomly distributed inclusions of the metals being examined.

The elements that can be detected by this technique depend on the energy of the X-ray beam focused on the sample. With an electron beam of 10 000 eV, zinc, copper and iron can be detected. Maps of these different elements can be created to compare the distribution of the metals in the tissue. For example, in X-ray fluorescence maps from the hippocampus, a region of the brain important in memory that is badly affected in Alzheimer’s disease, does the iron distribution vary across the cell population in the same way zinc does?

Choosing the spot size is also important. With a bigger spot size a larger area of tissue can be mapped, but many cell bodies in the brain are around 10 µm in diameter so choosing a smaller spot size may allow the metals to be pin-pointed to specific cells.

X-ray fluorescence mapping will detect atoms of an element no matter what chemical form it is in and no matter what other elements it is bound to. However, at Diamond the way the tissue absorbs X-rays can also be measured to provide information about what form a metal is in. For example is iron in the safer Fe3+ form that we expect the body to store it in? Or is there evidence for the more toxic Fe2+?

Without the intensity of synchrotron X-rays it would be extremely difficult to detect these metals while maintaining the spatial information. By studying the changes in iron and other metals in Alzheimer’s disease compared to people who died with a healthy brain, researchers hope to improve understanding of the disease that will hopefully lead to better treatment and ways of diagnosing Alzheimer’s disease.