Facilitator: I would like to introduce our two speakers tonight.
The first is Dr Dominic Hare, who holds an Australian Post Doctoral Industry Fellowship from the Australian Research Council, studying how commercial analytical technology can be used to image metals in the brain. He completed his PhD in Analytical Chemistry in 2009 and currently works in the Elemental Bio-Imaging Facility within the School of Chemistry and Forensic Science. His research interests are centred around integrating advanced analytical technology into systems biology. He collaborates closely with the Mental Health Research Institute in Victoria at the University of Melbourne helping to provide neuroscientists with access to imaging techniques otherwise not available to them.
Our second speaker is Dr Blaine Roberts and he is a Senior Research Scientist at the Mental Health Research Institute. His research interests are in the field of neuroscience, particularly in neurodegeneration. Dr Roberts completed his PhD in 2007 in Biochemistry and Biophysics from Oregon State University where he studied the neurodegenerative disease Amyotrophic Lateral Sclerosis also known as Motor Neurone disease. Currently he is investigating the role that proteins and metals have in Alzheimer's disease, Motor Neurone disease and ageing.
Dr Roberts' research is focussed on developing the tools to allow the direct study of metal containing proteins from biological tissues. These techniques are being used to unlock the previously enigmatic role of metals in biology and diseases.
So with that, as part of our National Science Week and the 2002 Ultimo Science Festival, UTS is proud to host this science public lecture titled, Trace Elements: As precious as gold for your health. Our first speaker will be Dr Dominic Hare.
Dr Dominic Hare: Good Evening everybody. Thank you. I feel like I'm about to give a sermon. So, thank you for the introduction, Phil, my name is Dominic Hare, as Philip said. I'm a postdoc here at UTS. I'm here today to talk to you a little bit about how analytical technology is being used to examine trace elements in health. Blaine's going to talk after me more specifically about how these trace elements are important in health and disease. So hopefully we give you a nice big overview showing how chemistry and biology are marrying very nicely.
So, to do things a little bit backwards. First I'd actually like to thank a few people who have provided assistance for this talk. Firstly, I'd obviously like to thank UTS. I've been at UTS for quite some time. I studied my undergraduate degree, honours, PhD, postdoc. It's been a great place to learn and develop. Now I work in the Elemental Bio-Imaging Facility which was founded by Philip in 2008. We've done a lot of great work since. Some of that work has involved close collaboration with the MHRI, where I first met Blaine. We've been working together for a few years now.
So I'm - as Phil said - I'm funded through the Australian Research Council, through the Linkage grant scheme which is a program that allows organisations to get involved in research in Australia through the Linkage scheme. Agilent Technologies and Kenelec Scientific are the two organisations I work with. So I wanted to acknowledge them as well and especially Agilent Technologies who have provided me with some support for this talk.
I'm going to take us back to some real basics about general science before we get in a little bit more detail. So, firstly when we're talking about the little bits and pieces that make people up, we need to go all the way back to the start of the universe. So, the universe is around 14 billion years old. What that means is all the pieces, all the atoms that are flying around us at the moment, that make us up are all about the same age. The only thing that changes - the thing that makes substances new and different is how these atoms are assembled.
So think for a moment about something like your house or your car that you drive, maybe even the UTS Tower - although that obviously looks like it was built in the '70s and is going to stay there. All these things are made up of building blocks that have been around since the dawn of time. The only thing that makes atoms different - or the only thing that makes something new was when atoms move around and change their structure. So that in itself is the essence of what chemistry is. Chemistry allows us to study change. Change is something that happens when something reacts.
So let's think of something simple like how this cigarette lighter works, okay. I'm not going to set anything on fire. Now, this cigarette lighter is a really good example of chemistry in action. We take a fuel, which in this case is something like butane, which contains atoms of carbon and atoms of hydrogen. We mix in some oxygen from the air and add a spark to provide some energy. Now, the atoms that make up that butane, and the atoms that make up the oxygen, they rearrange, they turn themselves into carbon dioxide and water and they give off a little bit of heat. That heat is what we can actually use as a tool.
Now the only thing that's happened here - these atoms haven't changed. The atoms are existing in exactly the same state but they've just rearranged themselves to form something new. So to get a little philosophical on you I want you to take a moment to have a look around at the people around you; the people sitting to your left, people sitting to your right. Now, you share something in common with everyone in this room. The little building blocks of life that make you up have been around since the dawn of time. The things that make up your neighbour, your friends, even politicians, they've all been around, they're all the same thing and they've been around since the dawn of time.
These atoms have seen our galaxies form and our planet coalesce. They've seen species come and go, all the way to making you who you are. They will continue on their way after we're gone. They'll continue to be part of the grass, part of the sky, part of new people on earth and part of the universe as a whole. They've experienced 14 billion years of progress, give or take a few leap years of course. So it's a good thing that atoms are inanimate and they don't actually know any of this is happening. Atoms are the simplest building blocks of matter. Now, physicists will try and tell you that there are smaller things but us chemists like to keep things simple.
Atoms are made up of very fundamental particles. We have protons, we have neutrons and we have electrons. Protons and neutrons, they exist in the middle of an atom. Electrons, on the other hand, they are whizzing round that middle of an atom. They are whizzing around that nucleus at around 2000 kilometres every second orbiting that nucleus much the way the planets orbit the sun. Now, that's the classical theory - version of atomic theory, which has been tweaked somewhat over the years. But this basic theory works quite well for what I'm going to talk to you about today.
So, the number of these protons, neutrons and electrons are what dictates something is. So, the number of protons is what determines what type of element we're looking at. Hydrogen is the lightest gas on the planet. It's also the lightest element on the periodic table. It contains only one proton. Uranium on the other hand, is the heaviest natural element on earth and it contains 92 protons. So the number of protons that makes up - or the number of protons we find in the nucleus of an atom is what gives us an atomic number, or what makes an element what it is.
Every proton has one single positive charge. Now neutrons, as the name would suggest, are neutral. They don't have a charge and the weigh about the same as a proton. These determine what isotopes of a certain element is going to be. Now this might get a little bit confusing but bear with me it's going to be important in a second.
Iron, for example, has 26 protons and that's what makes iron unique. However there are several different isotopes of iron with differing numbers of neutrons. Some have 28 some have 30 some have 31 some have 32. Each of these isotopes, it behaves exactly the same as any other isotope of iron. The only thing that's different is that there are a different number of neutrons. Iron 54 has 26 protons, 28 neutrons. Iron 56 has two neutrons less - still the same number of protons.
Now electrons, on the hand, they actually don't weigh much at all. They are only around 1/2000th the mass of a proton or a neutron. But these are the particles that chemists really care about, because they are what determines how something will react - and as I said before, we're really studying reaction we're studying change. So if an electron doesn't weigh much that doesn't stop them playing a really important role. That, as I said, that electron is what makes an element reactive and that's how we can actually view how it changes in nature.
So let's take for instance, the example of iron again. Now, there's a big difference between the iron we see in a lump of metallic iron and the iron that we have in a protein like haemoglobin that carries oxygen around our body. The only thing that's different between the iron in these two examples is how electrons are behaving. Of course this is just one atom of the many, many atoms that make up you - there are a lot of them. There is, seven with 24 zeros after it, of atoms in you right now. That's the average human, who weighs about 70 kilograms.
So, most of those atoms are only taken up by a couple of types. Most of the elemental composition of your body is made up of just a couple of different elements. We have 65 per cent of your elemental make-up is oxygen; most of that being from water. Ten per cent of your elemental make-up is coming from hydrogen which is found obviously in water as well, but lots of other molecules in your body. Carbon and nitrogen, making up 18 per cent and three per cent of your elemental make-up respectively are - they form the background of proteins which make up cells, make up tissue.
Calcium, which is the most abundant metal in the human body exists at around one per cent and is the major inorganic component of your bones. But if you add all that up you'll see that there's another three per cent of trace elements that aren't accounted for. Now, 21 elements out of the periodic table have been identified as having a key biological function. Another four have suspected roles. Where are they in this picture? Where are these three per cent of what we call trace elements?
So, I'm going to give you a couple of examples to talk about just how trace these things are. Now 0.1 per cent of the elemental make-up of your body are the elements sulphur, potassium, sodium, chlorine. These have a lot of different roles and by trace elements standards these are pretty concentrated, we would say. Now 0.006 per cent of your mass is iron, which is equivalent to around 4.2 grams. Now in a sense that's pretty much equivalent to the number of people we have here tonight compared to the entire population of Sydney. So, that's a reasonably small amount.
Now 0.001 per cent of your body is copper. That's the same thing that makes up electrical wires. Now, it accounts for around 0.072 grams of your body's make-up and that's about the same as this single grain of rice in five buckets of water. Now 0.000016 per cent of your body is iodine. That's the stuff that leaves your skin yellow when you use some type of antiseptic treatments like Betadine. It weighs around 0.02 grams and that's equivalent to one minute - so the time it takes for the big hand here to move one notch every six years.
Now, 0.0000021 - or did I leave one out - per cent of your body is cobalt. That's mostly in the form of something called vitamin B12 which you're probably all familiar with, but there are a few other things as well. It accounts for only 0.000003 grams. It's really quite hard for me to visualise just how small an amount that is, but we can compare it to one drop of water going into a volume the size of an Olympic swimming pool. So these are really really small amounts but just because there's not much there doesn't mean they're not important.
From a chemistry point of sense, if we want to measure how these things react we need to be able to look at them, but how do we measure something that is that small? Now, a really good way of demonstrating the principle behind this technology has actually been around for a while. I would have loved to have done it here but I have a feeling that it would have been a little difficult with the amount of fire that I'd need; a cigarette lighter's not really going to cut it. But it's the same principle that gives fireworks different colours.
So we're all familiar - we've all seen fireworks before. The principle here is that every element has a characteristic spectra it gives off when we expose it to a large amount of heat. Now on the left you'll see an example of what happens when sodium is placed in a Bunsen burner flame. A Bunsen burner flame burns at about 2000 degrees Celsius, it gives off a nice orange flame. You guys have probably experienced this if you've ever over boiled a pot of salty water. In the middle you'll see potassium which emits in the same type of flame a quite brilliant purple colour. On the right we have copper which in the presence of chlorine gives a green emission spectra.
Now, some of these emission spectrum might actually look very similar to us but if we measure them objectively we can actually see that they are quite different. We can use it to sort of fingerprint what a trace element is. So, how does it work? So as our sample gets heated up to that 2000 degrees, it gets broken down into just the constituent atoms that form the sample itself. They are all flying around independent to one another. So those electrons that are whizzing around our nucleus, they take in some energy from the flame, kind of like the way a kid would take in a lot of energy if they drink a lot of red cordial. They start going around faster and faster and they jump up to a new energy level.
What happens, obviously, when a kid who has had too much red cordial - he needs to crash and these electrons will do the same thing. They'll move back down to another energy level and they're going to give off some sort of characteristic radiation. That radiation fortunately is light. We can measure light and we see it as these different unique colours. Now there are a few fairly complex factors, depending on what type of light that these elements are going to emit, and why they're different from one element to another element. But trust me they're different for each element.
Here's an example of what we'd see. We can measure these unique spectra. We have an emission spectra for hydrogen, iron and mercury and we can measure where this light is being emitted to fingerprint elements. I think this is a really good example of - well I find - one of the most impressive and unique things that have been done in modern science, is that we've been using emission spectroscopy. For quite some time we've been able to get a good idea of what trace elements - or of what the composition is of far away celestial bodies, like stars and galaxies that are trillions and trillions of kilometres away from us.
We can actually get a really, really good idea of what these things are made up of by looking at their emission spectra. We've taken this technology into space; the Hubble Space Telescope has an emission spectrometer on it. Right, now I think one of the world's most ambitious scientific experiments, the Mars Science Laboratory on the Mars Curiosity Rover, right at this moment is measuring emission spectra on a world that's over 50 million kilometres away. So emission's great but it's also not ideal. The thing is, emission spectroscopy, as you can see, measures things from a distance.
It's like trying to count everyone in this room by only looking at their shadows. So isn't it a lot easier to actually - or a lot more reliable, to go directly to the source, the same way that you would have seen me earlier trying to count things on my fingers. I still do that to this day. So it's the same idea about using emission spectroscopy to look at what far away bodies in space are made up of. We can't be sure until we can actually go and take a sample and bring it back, if we want to be more accurate.
So to understand that we need to go back and revisit what I talked about, what makes up an element. What makes an element what it is. Now remember I said there are three distinct particles. We have protons, neutrons and electrons. Now if the only thing that's different about an element is the number of particles there is, how can we tell them apart? Everything weighs something and that's only become official since 4 July this year when scientists in France discovered the Higgs Boson because of course nothing had mass before then.
These small particles they are all the same; they have mass and these all contribute to what an element's mass is. Protons and neutrons weigh about the same, an electron weighs, as I said, 1/2000th of that mass. So, if an atom of phosphorous has 15 protons and an atom of sulphur has 16 protons, wouldn't it stand to reason that an atom of sulphur must weigh more than an atom of phosphorous? Okay, so how exactly do we weigh an atom? I don't have a set of tiny scales. I don't have really small tweezers so I can't put them on there.
We need to actually come up with a way to weigh something and in chemistry we use things called mass spectrometers. Now, the idea of a mass spectrometer was first conceptualised a century ago this year actually. It's resulted in more than a couple of Nobel Prizes being handed out. Mass Spectrometry itself is a fairly all-encompassing term and it's used to look at a series of different designs that all essentially do the same thing; they measure the mass of small molecules or elements.
For trace elements we used something called an Inductively Coupled Plasma - Mass Spectrometer or an ICP-MS for short, because we wouldn't be chemists if we weren't using acronyms. Now if a Bunsen burner flame burns at 2000 degrees Celsius, what would happen if we make that flame 10,000 degrees Celsius? That's hotter than the surface of the sun, and we have a whole lot of electrons flying around that we throw in as well. If a 2000 degree Bunsen burner flame can make an electron move to another orbital, surely a 10,000 degree flame can get rid of that electron altogether. We do that in an ICP-MS, or at least for a couple of milliseconds, which is all the time we need to make our measurements.
The great thing about having charged particles - if we get rid of an electron and an element has more protons than electrons so it has an overall positive charge - the great thing about having charged particles is that we can use electric fields to manipulate where they go. What we can do is using this supercharged Bunsen burner, we can make our charged particles and direct them into a mass spectrometer and make our measurements. So the design of an ICP-MS is not as old as the mass spectrometer itself, the first mass spec was built in, I think, was built in 1912 and it was nothing more than a few series of glass tubes and a pump, I think.
ICP-MS was first commercialised in 1983, which happened to be the year I was born. Unlike me it's gotten smaller. The original ICP-MS really caused - well it probably caused a lot of headaches for laboratory managers when someone came in and said I really want to buy this new toy, they'd go where are we going to put it. You really needed a room these things were quite big. Modern day ICP-MS and this is one that is housed over in Building 4 here at UTS is actually a lot smaller and it probably only takes up about a metre square of bench space.
But don't be fooled. That metre square of bench space will still set you back about the price of a small unit in somewhere not very good with - in today's standards no running water or a shared bathroom and things like that. They are still expensive. Now an ICP-MS has several distinct areas that we can look at. Here's a video just showing you what happens in an ICP-MS. We have - this is an older model, we also have here at UTS a - the sample which is usually in liquid form is aspirated and what that means is we have a stream of argon gas that forms some small droplets and these small droplets are filtered out using a fairly simple geometric spray chamber here.
The particles go through into our plasma and all that heat that we get from our plasma and the source of electric, it drives our sample down, gets rid of all the water. It decomposes it down to just the constituent elements the same thing that would be happening in a Bunsen burner. They get atomised but the additional thing is they also get ionised. What that means is that we're ripping those electrons off and getting our mixture of charged particles and neutral species where nothing's actually happened to them. These particles move into an interface region where they're going from atmospheric pressure to a vacuum.
We can use electric fields to direct our charged particles off axis into a hole going through into a mass spectrometer while getting rid of our neutral species, which we don't want. So we're again filtering things out. We're taking just the bits that we're interested in. These particles move into a quadrupole and this is what sorts them out. This is essentially what weighs them. We use oscillating magnetic fields to direct just the mass that we're interested in into a path which has a stable trajectory, while all of the other masses that we're not interested in - we can tune those electric fields so that just the particles we want go in and the rest go off into space.
Those particles then are detected where we actually convert physical matter into an electrical signal which we can see on our computer screen and we can use to do our interpretation. So, the thing that's really good about an ICP-MS is its sensitivity. It's been having a bit of a renaissance of late. While I said that it's been around since 1983, the biological community has probably really only appreciated what it's capable of in the past ten years or so. Improving technology, as well as better awareness from biologists has given us a tool that's capable of measuring down to those tiny levels that we need to see, when we want to look at how trace metals change in health and disease.
In fact in the right laboratory, in the right conditions a modern ICP-MS is able to detect down to one part per quadrillion, which is equivalent to one centimetre and 50 round trips to the sun. That's 15 billion kilometres. Think about that, one centimetre every 15 billion kilometres. That is how sensitive this equipment is. So you can see why it's quite useful. However, one of the limitations is that it's a little bit unspecific. I mean, as I said in the video, we have to put our samples in as a liquid. We take our tissue that we're interested in, we dissolve it in acid and into the machine it goes.
But a biological system is much more complex than that. If we think about what makes up a biological system. We have our atoms, our atoms form molecules, those molecules form proteins, those proteins form organelles they are the factories of cells - those cells of which there are 50 trillion in your body. Those cells which make up tissue, they make up organs and all that goes together to make you, you. So when we want to look at trace elements we can see that we have to go back several steps to be able to identify these changes. So what do we do if we want to look at these diseases at the source? How do we pinpoint small changes, so molecular or cellular changes?
Now, grinding up tissue just doesn't necessarily cut it. What we do use is we use lasers. They're not big and exciting lasers, they don't make much of a noise, we can't see them, but they do exactly what you think a laser would; they burn stuff. We fire tiny laser beams, as small as 100th of the width of a human hair onto a biological sample. So, in my case I usually look at the tissue the cut tissue sections. But we can look at teeth; we can look at all sorts of stuff, even a human hair it's small enough. This discreetly allows us to sample a tiny area.
While I spent most of my PhD and the other research I work in now doing, is working out how to take those small areas of analysis and actually turning it into images. So we can drag this laser beam across a sample much like the way that a printer prints out a picture that comes out of your computer. We can direct this laser beam across a sample and actually use this to produce images. As the laser moves across the sample it oblates, it vaporises some of our sample and that sample is light enough to be carried by a carrier gas through to our ICP-MS.
Our sample goes into the ICP-MS, it gets ionized, it gets separated according to its mass. We take that signal, we put it in our computer, we do some processing, take it away, apply a few algorithms to it and we can turn those tiny pinpoint laser beams into images that look at distribution of trace metals in tissue. The beauty is it allows us to look at really small areas without having to cut them out. Which even if it were possible to look at the small areas we want to, it's fraught with problems. When we talk about trying to look at single cells, you can't exactly get a knife in there and cut out a single cell. It's fraught with problems.
We have to use nasty chemicals, there's a contamination issue, and in some cases it's just not possible to do. Here you can see what we would call a healthy mouse brain. It's been sectioned through the middle. It allows us to look at where brain iron is concentrated. In this case red means a lot and green means not much and blue means very little. Here's another mouse brain that this time has been treated with a drug that's used to model the effects of Parkinson's disease. This technique allows us to look at how changes to the really small region of the brain that's affected by Parkinson's disease.
So, how changes in those trace metal concentrations can be viewed in these animal models, and how we can use them to help look for new - reasons to explain why the disease is happening - and new ways to target them. So Blaine's going to talk about trace metals and disease in a moment. Scaling the technology up we can also use this imaging method to look at how we can reconstruct trace metals in the mouse brain. Now, why the mouse brain you ask. Mice are still one of the most commonly used animal models in disease research. Now, rest assured we're not using animals to test shampoo or test cosmetics. In Australia scientists are held to a very, very high standard of animal ethics.
But mice remain one of the most important factors in developing new therapies for disease or trying to treat disease or understand the process behind disease; everything from heart disease to Alzheimer's disease. So using that same data we can construct these two dimensional - or we can use these two dimensional images to build 3-D models. They actually look at very distinct regions of the mouse brain and how changes within these areas in disease models, how they can be isolated and how they can be studied and how we can actually look at them for developing new therapeutic strategies.
So, the beauty in this technology about everything that I've shown you so far, is that we can look at localisation of metals in specific tissues and now we're approaching the single cell. But this only tells part of the story about trace elements and health. Moving forward ICP-MS and analytical technology as whole, is probing deeper and deeper into living systems.
Now, this would be a perfect opportunity to introduce my colleague Dr Blaine Roberts. So this idea that I've introduced about us being made up of - sorry, the healthy us being made up by a bunch of different atoms, it would then stand to reason that a diseased us is going to be made up of those atoms as well. They're just going to have changed somewhat. Blaine's going to talk about this now.
I thank you very much for your attention.
Dr Blaine Roberts: Thanks Dominic. Thanks to everybody here showing up and giving me a chance to tell you about some trace elements. So, as Dominic was talking about that last figure where he has - I really liked that figure actually where you start off with pictures of people and then breaking it all the way back down to atoms. So Dominic was looking at how metals are distributed throughout a whole organ. What I'm going to talk about is one step smaller again. So I'm going to look at how metals are involved with proteins.
So again the periodic table you can see 100 or so elements, 114 or so, most of them are quite - some of the larger ones are quite exotic. For the most part we're made up of the top half of this periodic table as Dominic had mentioned. We're not just a random selection of these elements. If you look at their distribution throughout the universe you could see that the most abundant ones are hydrogen and helium mostly from stars. Then the third most abundant one is oxygen, which is a good thing because we need that to breathe and to make energy and things.
Then not too far down the list is iron which, as many of you know, iron is very abundant in the earth's crust. I don't know if - in the States we always talked about - at primary school and things like - maybe the centre of the earth was made of peanut butter because we really enjoyed peanut butter. I don't know if it's Vegemite here but anyway the centre of the earth is actually of iron. It's one of the most abundant elements here on the planet. So, to go from elements on up the chain a bit of how these molecules are, what are the basic biological elements of life? So how do these things come together and what do they do and what is important.
So for living organisms, there are basically four categories. Lipids, you have proteins, carbohydrates and DNA. I have a couple of pictures of nice examples representing it. Lipids - olive oil. I really like olive oil. I would have put a picture of a - there was actually a nice picture of butter that was starting to melt in the pan but I said well maybe that's not so appropriate since we're talking about health and we want everybody to be healthy, we should be eating more olive oil less butter.
Then we have our carbohydrates, the nice breads you dip in your olive oil and then I can never go past a nice T-bone steak. I grew up on a farm so grilling - it has to be grilled on charcoal, it can't be gas, right, you've got to do that on charcoal to have a nice T-bone steak. Then you have DNA. DNA I find is wonderful but as a protein chemist it's a little bit boring for my taste and I'll just go over the central dogma of biology. Essentially, you take DNA, which is very important; it's our code, it tells a cell what to do essentially. But you have DNA goes to - is transcribed to RNA and then that RNA is turned into protein. So DNA really is just the code to tell you what proteins to make.
Those proteins then go on to do functions and you have carbohydrates which produce your energy and lipids which are incredibly important. Lipids define actually what a cell is. So on this image here you can see - you have an image of a cell - or a cartoon of a cell, and then the nucleus in the middle. Those membranes are really what make those compartments. Those membranes are also incredibly important in producing energy because they allow the carbohydrates to be burned which allow chemical gradients to be made. They allow separation to occur and then those chemical gradients are made to - are used to make ATP which then lets us move and think and do.
This is a bit of my - one of my favourite quotes and it's pretty much anything a cell does a protein does it. So, my area of interest is in proteins and it's because whenever we move, or think or when an amoeba engulfs a bacteria, it's proteins that are doing it. They are doing the work. Here is just a - and they're pretty as well. So proteins when you solve their structures down at the atomic level what you can see is they have a lot of motifs and they've been named. Like this one here actually has a Greek key motif that maybe many of you will recognise because it's used a lot of times in decorating and things.
But then also this enzyme is one of my favourite enzymes as it has a copper and a zinc in it and it's one of the most common enzymes throughout the kingdoms of life. So, the human genome has been sequenced and there are approximately 22,000 genes in the human genome. Each one encoding for a unique protein and this here is just a graph demonstrating their structural or their functional categories of how these proteins work and what do they do. The oxidoreductase which is over on the right hand side there - sorry the pointer doesn't work against the LED screen here - but the oxidoreductase class, that's where that copper zinc SOD, or superoxide dismutase protein falls in.
But you can see there's a huge section in the middle about 23/24 per cent of proteins we just don't know what they do yet. So there's a lot left to learn, we're very far from knowing everything. One of the interesting parts about proteins is that about 30 per cent to 50 per cent of them use a trace element like copper, iron, zinc, for their function. So those elements are required for function. Our current technology - when we study proteomics or study proteins - lacks the ability to detect those metals in proteins. They look just at the protein itself and they miss the elements like copper, iron and zinc. Some examples are very important.
Metalloproteins some that you've heard of as haemoglobin which Dominic showed the structure of earlier. Obviously it's vital for moving oxygen around through our body. Ferritin, which is a protein that's involved in storing iron. Matrix metalloproteinases, which have many roles. One of their roles where things go wrong is often they can be used in angiogenesis of cancers, which is the metastasis which is a very bad thing. You've seen a lot of these metals and heard of them, or these minerals.
If you look at the back of your vitamin and mineral supplement which I imagine many of you take because you're here because you want to know about trace elements and health. You can see this long list of your vitamins and your minerals. You have vitamin B12, which we know which has cobalt in there which is very little in the body but it's very important. Then you have zinc, selenium, copper, manganese, and chromium.
Who knew that there's a recommended daily allowance of chromium. Most people from my era at least, we know of chromium from the movie Erin Brokovich where she goes around and has the big law suit about chromium being toxic. But it's also required for life. There's actually in very special circumstances you can make individual's chromium deficient and it seems to have a function in insulin signalling and energy. You go down the list and some of them they don't have a recommended daily value but nonetheless we know we need them and some of them we know how much you need in order to be healthy.
But after that we don't understand what they do in disease and often what happens in normal biology. So what proteins require Molybdenum, what proteins require chromium, manganese and things; those are questions that we're only just starting to answer. So, in disease cases there are a number of classical examples. I've just picked a couple where elements - particularly iron and copper - manifest themselves and there's often genetic links or they could be dietary links. I imagine many of you are familiar with becoming anaemic where you don't eat enough steak potentially.
You have other diseases like hemochromatosis and this here is a cartoon describing the primitive technique of trying to cure hemochromatosis by doing bloodletting. So you get a lot of iron out of the body by letting out your blood. Then you have Wilson's disease, which affects a large number of your organs. So it's never a single organ that's just affected, it's usually the entire organism. You can see in the diagram here where you see liver, kidney, heart, and your eye. Menkes disease which is involved with copper and again it affects a large portion of the organism; particularly one of the parts I'm interested in is in the brain. So it affects your memory and you have a sense of vertigo and things like that.
Another way that for - in healthy states is malnutrition, where you just aren't getting enough. We've heard classic examples with other vitamins like vitamin C and things. With the Captain Cook, where he got all of his sailors to eat dried limes I believe it was, to help prevent scurvy and that was because of a deficiency in vitamin C. So by eating the limes and the lemons they prevented the symptoms of scurvy because they have enough vitamin C and vitamin C is required for tissue maintenance and things. Where you can also have - zinc can also become a deficiency. As far as minerals goes, zinc is by far the biggest deficiency in the world.
There are approximately two billion people that are believed to be deficient in their daily intake of zinc, this has consequences on wound healing and immune function, and also there are developmental consequences for the adolescent children and developing children and their ability to grow and neurological things. Then also there's interesting studies on linking deficiency in zinc and behaviour. There are actually studies in prison populations where they show that aggressive prisoners, when they were supplemented with zinc actually their aggressive behaviour decreased. So it's not necessarily that just zinc does it but it is interesting that it does - can affect the behaviour.
So one of the things you heard about in the introduction is that I study - I'm a neuroscientist which basically that just means I look at neurons. A lot of people try to define well what is a neuroscientist, how do I train to be a neuroscientist. A neuroscientist - basically, the common thing is that you are looking at a neurological tissue or cell type and after that all bets are off. You could be a mathematician who does neuroscience. You could be a biophysicist, a biochemist, material scientists, analytical chemists - a little bit of everybody gets in on the neuroscientist type of stroke.
But one of the diseases I look at is neurodegeneration and in this case Alzheimer's disease which is the most common form of dementia and I'm sure everyone in here has heard of before. It was discovered in 1906 - not necessarily discovered, but first described - in 1906 by Alois Alzheimer. As is the case in history usually the second or third person gets all of the credit - because it had been discovered previously - but he was the champion and the salesman who convinced the world about Alzheimer's disease. This is a picture here of Auguste Deter who was the case study that he used to describe the disease.
Alzheimer's disease is an impending epidemic and you hear things about it in the news. As the population ages, there's increasing numbers of people. By 2050 the estimate is that there will be over 50 million people suffering from Alzheimer's disease and, besides being a devastating disease that robs us of our consciousness, essentially - or what makes us human - it takes a very long time, eight to 20 years and it costs a lot of money. It's projected to cost 20 trillion dollars over the next 40 years.
One of the things with Alzheimer's disease is to diagnose the disease it's always probable Alzheimer's disease until autopsy. So it's a diagnosis of exclusion. We know you don't have this, this or this so it must be Alzheimer's disease and it can't really be confirmed until autopsy. Here you can see a comparison of a healthy brain which you can see is nice and plump compared to an advanced Alzheimer's diseased brain and you can see the increased vacuolization and just the general atrophy that's occurred in the brain.
Approximately 50 per cent of the neurons are lost and there's these pathological hallmarks called amyloid plaques, which are formed up of these particular proteins and these plaques define if you had Alzheimer's disease or if it was a different type of dementia. Dementia of Lewy Bodies et cetera. Iron, copper, zinc, potassium, rubidium and other trace elements are altered in an Alzheimer's diseased brain and one of the things we're trying to do is figure out how do these elements change and how do they relate to the disease process.
A second disease that - neurodegenerative disease - I study is Amyotrophic Lateral Sclerosis or Lou Gehrig's disease, who was a famous baseball player in the United States; played baseball with the New York Yankees with Babe Ruth and, if anybody's a baseball fan it's also called Motor Neurone disease, or it falls under that classification. It's a disease that specifically it affects motor neurons and not other types of neurons in your body. That results in your inability to walk and eventually it moves up the spinal cord and you lose your ability to move your hands and eventually to - often death is either through - inability to breathe.
Now motor neurons are quite specialised and it's very interesting, we all have plenty motor neurons of course, so we're born with more motor neurons than we have today. But there's motor neurons that actually come out from your lower spine and go all the way down to your big toe and allow you to move your big toe. So they're over a metre long which is an impressive feat. One of the genetic causes of the disease is actually mutations in that enzyme I showed earlier, copper, zinc, superoxide dismutase. So one of the things we're trying to do is figure out how does copper and zinc affect that enzyme and how does that relate to the disease process.
Here I'll just show you - there's a bit more chemistry on this slide than you need to look at - but basically we have the purified protein. So if you take this protein, the copper, zinc SOD and you purify it down it is a beautiful green colour. If the only thing you do is remove the zinc it goes to a pretty blue colour. One of the wonderful things of working with metalloproteins is that they are almost always coloured, like haemoglobin's bright red. But in this case, just the loss of that zinc dramatically changes the function of the enzyme.
So, instead of the enzyme being beneficial and actually scavenging superoxide - which is the most common oxidant that we encounter - instead of it being protective and being antioxidant, so working like vitamin C would, it actually works backwards and actually produces superoxide. We think that's the major cause, or one of the major causes, for the disease. So how are we going to measure just the metal status of those proteins and we're going to link this back again to the instrument that Dominic introduced earlier, which is the Inductively Coupled Plasma Mass Spectrometer.
Now where Dominic uses a laser to figure out how the elements are distributed throughout an organ, I use an instrument called a High Performance Liquid Chromatography - or an HPLC, because again we're chemists so we like our acronyms. So we use an HPLC, ICP-MS. If we can get a couple more in there we'll have a new alphabet. But basically what we do there is, we can take a little bit of that tissue and we can extract the proteins out of it. We can pass them into the ICP-MS, and if you remember from the diagram it breaks it all down into its elements. We can monitor the amount of iron, copper, zinc, manganese, cobalt whatever element we're looking at.
At the same time, using that HPLC technique we can figure out how big those proteins are and proteins are very much like us, that we come in all different sizes and so do proteins. The size of the protein tells us a little bit about it and we can make guesses potentially to their function and then also monitor how the protein changes in the diseased state. So again here's just an example showing a number of proteins, metalloproteins that we can measure. You can see from really big proteins like thyroglobulin to actual small molecules like vitamin B12, which is cobalt in the main or has cobalt there. Then you have copper, zinc SOD which is the one we were looking at, and ALS.
So this measurement of metals bound to proteins is called metalloproteomics. Maybe some of you have heard of Omics type technologies. That's kind of out of the era of genomics which was looking at the genome. Then you have proteomics which is looking at the proteome. This is just a variant of it where we're looking at the metals associated with those proteins. Here's the application of metalloproteomics to several neurodegenerative diseases. One we have ALS, or motor neurone disease, which we talked about before.
We can see large changes between the copper, zinc SOD and some of the other copper containing proteins in the diseased tissue. We have Alzheimer's disease where we're seeing big changes in specific metalloproteins and Parkinson's disease again, which is linked with - has a long history of iron being involved in Parkinson's disease. We can see changes in the Parkinson's disease models as well. So that's what we're investigating and where we're going in trying to understand these diseases.
One of the things that we also do is using that same technique we can make these prettier pictures instead of just a squiggly line across the page. You get these beautiful maps that remind me of the - if you've seen the images of the ocean floor where you see these islands and volcanoes rising up from the ocean - you can see here where each one of those islands is actually a different zinc protein. So as we talked about 30 to 50 per cent of proteins having a metal cofactor we should see a lot of proteins if there's 22,000 of them in a cell. You can see here we can see a large number around 30 or so.
So, if you guys are interested, I just want to point out this webpage here, which is the micronutrient information at the Linus Pauling Institute. So if you have any questions, or you want to know more about the vitamins and minerals that you take particularly this is a great resource that has a lot of references, and really good descriptions of how vitamins, as well as, minerals are involved in human health and disease. There's even the Linus Pauling Institute prescription for health. They have recommended levels that you should be taking for vitamin C, et cetera.
Just to summarise, proteins are important for cellular function. As I said pretty much anything a cell does the protein does it. Metals are important for protein function. So proteins can't carry out their function without metals. Deficiencies can result in a number of diseases however we know very little about how these minerals, or metals, are involved in the disease. Particularly how they are involved with the proteins and that's what we're trying to get at. Our main question is how are metalloproteins involved in normal biology and also in Alzheimer's disease and in Motor Neurone disease.
So, with that I think we can take any questions, comments. Thank you.
22 August 2012
Metals play key roles in over 40 per cent of the proteins in our body, yet their involvement in health and certain diseases is still not clear. Some trace metals that are essential to life can be deadly if the delicate balance in the cell is disturbed. The ubiquity of trace elements presents numerous challenges to studying them within biological systems.
As our population ages, there is more and more urgency in studying how trace elements are involved in neurodegenerative diseases, like Alzheimer’s and Parkinson’s. This talk will examine how cutting edge analytical technology is providing new insight into how we can probe the role of trace elements in normal physiology, and how it is being applied to studying devastating diseases in humans.
Test Tags: medical science, trace elements
About the speakers
Dr Dominic Hare holds an Australian Postdoctoral (Industry) Fellowship from the Australian Research Council, studying how commercial analytical technology can be used to image metals in the brain. He completed his PhD in Analytical Chemistry at UTS in 2009, and currently works in the Elemental Bio-imaging Facility within the School of Chemistry and Forensic Science. His research interests are centred around integrating advanced analytical technology into systems biology. He collaborates closely with the Mental Health Research Institute at the University of Melbourne, helping to provide neuroscientists with access to image techniques otherwise not available to them.
Dr Blaine Roberts is a Senior Research Scientist at the Mental Health Research Institute. His research interests are in the field of neuroscience, particularly in neurodegeneration. Dr Roberts completed his PhD in 2007 in Biochemistry and Biophysics from Oregon State University where he studied the neurodegenerative disease amyotrophic lateral sclerosis (aka motor neuron disease). Currently, he is investigating the role proteins and metals have in Alzheimer’s disease, motor neuron disease and ageing. Dr Roberts’ research is focused on developing the tools to allow the direct study of metal containing proteins from biological tissue. These techniques are being used to unlock the previously enigmatic role of metals in biology and disease.
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