5-Aminolevulinic Acid
|
 |
It does sound it, but 5-ALA has a far more serious purpose than serving as an entertaining beverage on a Saturday night out. It is currently used in fluorescence-guided surgery on numerous types of cancers, including aggressive brain gliomas called glioblastomas due to its useful property of fluorescing bright pink when placed under ultraviolet light.
So the pink drink isn't actually pink?No, it's colourless. But it called the ‘Pink Drink’ (sold under the name Gliolan) because of the way it makes brain tumours glow bright pink under UV light, while healthy brain tissue remains normal in colour. Bright pink- that looks like it would be quite noticeable…Exactly! One of the main challenges of tumour surgery is ensuring no residual tumours are left behind, which could cause the cancer to resurface. This is especially relevant with high-grade glioblastomas, which are the most common and aggressive type of cancerous brain tumour in adults, with a prognosis of only around 15 months. Fluorescence-guided surgery helps surgeons accurately identify cancerous cells, enabling the removal of as much of the tumour as possible. Given the aggressive nature of glioblastomas, the goal of this technique is to improve a patient’s prognosis rather than achieve a complete cure. |
 Brain tumour cells fluorescing bright pink after 5-ALA ingestion before surgery. [Photo: Universitätsklinik für Neurochirurgie, Inselspital Bern, CC BY-NC 4.0] |
This shows that the amino group (NH2) is bonded to the 5th carbon in the chain, as shown in the diagram.
 |
 |
|
| 5-Aminolevulinic Acid (5-ALA) [Image: Wesalius, Public domain via Wikimedia Commons] |
Spacefill model |
To fully understand the process of precisely illuminating cancerous cells from drinking a pink solution, we have to go on a biochemical journey. This journey starts with 5-ALA’s main role in mammals: producing haem.
Yes, haem (MOTM for February 2006), sometimes spelled 'heme', is a molecule that forms part of the protein haemoglobin, and one of haem’s main purposes is forming the oxygen-binding site in haemoglobin. Haem is also found in proteins associated with electron transfer in oxidative phosphorylation, the final step in aerobic respiration, which is needed to produce ATP (MOTM for January 1998) — the main cellular source of energy for all our bodies’ cells. This makes sense as to why our brain cells would need to produce haem.
That is a very good question, and it actually involves cunningly exploiting a small difference in the haem-synthesis pathway in cancerous cells.
The synthesis of haem is a multi-step pathway, involving 8 different enzymes. Most of the intermediate reactions occur inside the cell cytoplasm, with the final steps occurring inside the mitochondria. The complex, multi-step synthesis of haem, shown in the diagram below, begins with a reaction that synthesises 5-ALA from succinyl coenzyme A and the most basic amino acid, glycine (MOTM for April 2010). In plants this occurs by a different route, shown in the blue dashed box. From there, the 5-ALA is converted into a pyrrole ring molecule (PBG), and then a series of enzymes join a number of these rings together to eventually form protoporphyrin IX (PPIX), shown in the red box.
The complex, multi-step synthesis of PPIX, a precursor to haem.
[Image: Crwoodford4, CC BY-SA 3.0 via Wikimedia Commons]
The molecule that's produced from this complicated process, protoporphyrin IX (PPIX), is a type of organic molecule called a porphyrin ring that consists of a fully conjugated system (one which contains delocalised π-electrons). This makes the cyclic ring very adept at donating electrons, which is a fundamental component of both aerobic respiration and photosynthesis.
 |
|
| Protoporphyrin IX (PPIX) [Image: Fvasconcellos, public domain via Wikipedia Commons] |
To some extent yes. In some plants, chlorophyll (MOTM for May 2000) can be synthesized via the same pathway as haem, sharing common steps up until PPIX. PPIX is harnessed all across the biological world for its capability to form metal complexes, which are structures that contains a central metal ion bonded to surrounding ligands. These ligands then donate their lone electron pairs to the metal species, forming coordinate bonds.
These complexes are fundamental in the natural world due to their ability to transport or store molecules and other particles. The ability for transition metals to also have several stable oxidation states is key in allowing complex biological systems to function, which all fundamentally rely on the standard interactions between charged particles.
 |
 |
| Chlorophyll b - the green pigment found in plants. [Image: Yikrazuul, public domain, via Wikimedia Commons] |
Haem B - the red constituent of blood. [Image: Yikrazuul, public domain, via Wikimedia Commons] |
True. Anyway, back to producing haem! The final step of the haem synthesis pathway involves the insertion of the Fe2+ ion into the porphyrin formed thus far, which then holds the ion in place with 4-coordinate bonds provided by the lone pairs of the nitrogen atoms in the ring. Other co-ordinate bonds are then formed with polypeptide chains or oxygen, which helps form the functional protein. This process is catalysed by the enzyme ferrochelatase, which is an intra-membrane enzyme found on the matrix-facing side of the mitochondrial inner membrane.
The final step in haem production - adding Fe to the centre of the porphyrin ring.
However, the key point here is that research has suggested that the gene that codes for ferrochelatase, FECH, is less effective at producing the enzyme in glioblastoma tissues compared with normal brain tissues. This then leads to the accumulation of protoporphyrin IX in cancer cells as there is a far slower rate of production of haem.
Yes! Exactly that. Since there is now a large difference between concentrations of PPIX in healthy brain tissue and tumour cells, the tumour cells can be distinguished from normal cells using ultraviolet light of wavelength 410 nm, which is absorbed by the molecules of PPIX. Some of this energy is then used up to make the molecule vibrate and rotate, but the remainder is re-emitted as fluorescence. Because the emitted light now has less energy, it will have a slightly longer wavelength of 635 nm, which corresponds to a red colour (which looks more pink if it's not very intense). So you shine UV light onto the sample, and the cancerous cells, and only those, will glow pink!
With 5-ALA’s help, surgeons can successfully remove the whole tumour in 70.5% of cases, compared to 30% of cases when 5-ALA isn’t used. This is likely due to glioblastomas having threadlike tendrils which extend into surrounding areas of the brain. Under normal light, it can be difficult to identify the edges of the main part of the tumour. This means surgeons would have to leave more of the tumour to make sure they do not remove healthy tissue with the consequent effects on patient brain function. By taking the Pink Drink before an operation and turning on a violet light, the edges of the tumour are more clearly visible, meaning more of the tumour can be safely removed.
Are there any other uses for 5-ALA in this area?As a matter of fact, yes. 5-ALA can not only be used in fluorescence-guided surgery to highlight tumours but can also be used in Photodynamic Therapy. This is where light of wavelength 635 nm is directed at the tumour, killing the malignant cells instead of just making them fluoresce. In contrast to fluorescence-guided surgery, photodynamic therapy can also trigger an immune system response, where cells of the immune system are attracted to the site of the tumour and clean up the debris. Okay, well how does that work then?This acts in a similar way to how PPIX fluoresces, but acts slightly differently. If red 635 nm light is directed at the tumour, the PPIX molecules absorb it and this time the energy is given to an electron which is excited to a much higher level than before, to a so-called 'triplet state'. Once in this excited triplet state, this excess energy can be transferred from the PPIX to other nearby molecules, such as oxygen. This then ‘excites’ an electron in the O2 molecule, making it very reactive and unstable. This can then react with other oxygen molecules, or with water, to form multiple reactive oxygen species (ROS) such as hydrogen peroxide (H2O2, MOTM for Sept 2006). These ROS then have the capability to react with other biological molecules inside the cell, such as lipids and DNA, causing damage to important metabolic processes which will eventually trigger cell death. |
 Photodynamic therapy procedure. Patients are injected with a photosensitizer (such as 5-ALA) which is selectively retained by cancer cells as compared with normal tissue. Doctors then use fibre-optic probes to expose the cancer to laser light. This activates the photosensitizer and produces a toxic reaction that destroys the tumor without damaging the surrounding normal cells. [Photo: John Crawford (Photographer), Public domain, via Wikimedia Commons] |
Mass tumour cell death triggers inflammation, which releases molecules called cytokines, which then attract macrophages (a type of immune cell). These macrophages then “engulf” any foreign particles from the tumour cells and “wear” them on their cell surface membrane. This then allows other immune cells, such as cytotoxic T cells, to recognise and destroy any remaining malignant tumour cells.
Similar to fluorescence-guided surgery, patients with high-grade glioblastomas first ingest the ‘pink drink’ containing the 5-ALA before the surgery. Then, once surgeons have access to the area of brain tissue that contains the tumour and have cut away as much of it as possible, a beam of red light with wavelength 635 nm is shone in the area. This will then trigger cell death in the tumour cells that contain the excess PPIX, helping to eradicate much of the remaining cancer tissue.
Wikipedia: Protoporphyrin IX; 5-ALA; Photodynamic therapy
Back to Molecule of the Month page. [DOI:10.6084/m9.figshare.29222675]
