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Aminolipine: formaldehyde-free substitute for preserving biological tissues

For almost 125 years, undertakers as well as physicians in the fields of anatomy and pathology have used formaldehyde to preserve biological tissue and whole cadavers. Yet, formaldehyde is highly toxic and linked to cancer. A team led by Prof. Dr. Bernhard Hirt from the Institute of Clinical Anatomy and Cell Analysis at the University Hospital in Tübingen has developed a formaldehyde-free replacement substance. The researchers are now planning to market this substance.

Twelve operating tables are arranged in two parallel rows in a semi-circular room on the ground floor of the Institute of Clinical Anatomy and Cell Analysis in Tübingen. The bluish light from the fluorescent lamps bathes the scene in a cool and sterile atmosphere. Non-anatomists would certainly not suspect that cadavers lie beneath the blue sheets covering the tables almost down to the floor. There is no smell of decay or chemicals.

Bernhard Hirt, medical director of the Institute of Clinical Anatomy and Cell Analysis, reaches out and touches the head of a cadaver underneath the blue sheet. He seems to be able to easily move the head upwards. Later on, surgeons will be trying out the latest surgical techniques on the donated cadavers. The cadavers on the twelve tables have been preserved with a new fixative called aminolipine, which has been developed by Hirt’s team. The solution prevents cells from self-digesting and halts microbial putrefaction and decay.

Glossary

  • Being lytic is the feature of a bacteriophage leading to the destruction (lysis) of the host cell upon infection.
  • The toxicity is the poisonousness of a substance.
  • Pathology is a section in medicine which deals with the reasons, the development and the consequences of pathological symptoms as well as of abnormalities.
  • Fluorescence is the spontanous emission of light in a certain wavelength after excitation of a molecule with light of another wavelength.

“Formaldehyde fumes have a strong smell and irritate the eyes”

Prof. Dr. Bernhard Hirt with aminolipine (in the glass bulb on the left) and the two starting substances. © Braitmaier

The scientists from the Institute of Clinical Anatomy and Cell Analysis are organising dissection courses for around 400 future physicians. The cadavers need to be preserved using a highly toxic formaldehyde solution to prepare them for teaching purposes. Formaldehyde is currently the only approved agent for the long-term fixation and preservation of whole bodies. In contrast to tissue preserved with aminolipine, formaldehyde-treated tissue looks unreal and has an unnatural feel. The cadavers look greyish-brown, the muscles are extremely rigid and the joints are difficult to move. “Formaldehyde-treated corpses emit a pungent vapour and your eyes start watering shortly after exposure to it,” says Hirt.

The aminolipine project is coordinated by Prof. Hirt and was more or less born out of necessity. In 2006, the International Agency for Research on Cancer, IARC, classified formaldehyde as a carcinogen. In 2014, the EU followed suit (1, 2). As a result, the permissible workplace exposure limit for formaldehyde, which evaporates easily at room temperature, was lowered in Germany (3).

"We have a relatively big ventilation system in our building, and this is just about able to keep formaldehyde values below the permissible limits. However, other universities had to postpone their anatomy courses because they could not keep the levels below the stipulated limits. In 2024, the approval of formaldehyde as a fixative will be reviewed under the European Biocidal Products Regulation, and may lead to further proposals for restrictions.

Disinfectant with unexpected effect

Human cadavers two months after formaldehyde fixation (left) and aminolipine fixation (right). The cadavers were stored at room temperature. © Manfred Mauz, Institute of Clinical Anatomy and Cell Analysis

In order to be prepared for further restrictions on the use of formaldehyde, a team of biologists, chemists and physicians led by Hirt set out early on to find a substance that can replace formaldehyde and is less harmful to human health. They found what they were looking for in a disinfectant mixture that is used in hospitals and medical surgeries for disinfecting surfaces and instruments. The fact that the agent not only kills bacteria, viruses and fungi, but can also preserve tissue, was previously unknown to experts.

Hirt's team subsequently developed a new manufacturing process that has made it possible to selectively synthesise a specific ingredient in the disinfectant to 98 percent purity. "Tissue preserved with the original mixture was not ideal in terms of the feel and had an unnaturally greenish colour," says Hirt explaining why they decided to focus on one particular ingredient rather than on the whole mixture.

The researchers from Tübingen eventually came up with aminolipine, which can be produced from coconut oil and an amino acid derivative. They were able to show that aminolipine destroys the three-dimensional shape of proteins and thus neutralises the body's own enzymes as well as microbial enzymes that would normally decompose the tissue. In the meantime, a patent application detailing the manufacturing process and the mode of action of aminolipine has been filed.

Standing in his office, Hirt holds a glass bulb up to the light. The aminolipine sample inside it shimmers pearlescent and has the consistency of wax. "It was not easy at all to produce a substance that was like formaldehyde in all aspects,” Hirt admits. "Formaldehyde penetrates tissues incredibly well. This is because it's a small molecule. It is also omnipresent in the air. There is no way to avoid being exposed to it,” says Hirt laughing.

Final steps on the path to producing a marketable product

The fixation solution is prepared by atomising aminolipine and bringing it into solution. © Peter Neckel, Institute of Clinical Anatomy and Cell Analysis

The researchers therefore decided to develop a new formaldehyde-free aminolipine fixative that is nevertheless able to reach the remotest corners of the body via the blood vessels. Another good reason for choosing aminolipine was that the researchers could not detect any aminolipine molecules in the air that could have affected human health. In addition, Hirt's research group ensured that aminolipine remains soluble at temperatures around freezing point, which is the temperature to which bodies are cooled prior to preservation.

The efforts have paid off: Hirt is set to receive 4.5 million euros in funding from the GO-Bio programme run by the Germany Federal Ministry of Education and Research. This three-year financial injection will help Hirt and his colleagues to develop aminolipine into a marketable alternative to formaldehyde. The researchers will also be using the funding to develop optimised product formulations that can be used to preserve any type of tissue, be it brain, pancreas or intestine. For pathological applications, the researchers will have to change the composition of the agent so that it can be used to stain and analyse the cell components.

The scientists from Tübingen are also planning a multicentre efficacy study in which four anatomy institutes from Germany and China will study in detail how well aminolipine is suited as a fixative and preservative. Last but not least, the researchers will have aminolipine tested for carcinogenicity, toxicity and environmental compatibility by certified external laboratories so that they can then apply for substance and product approval under the European Biocidal Products Regulation.

Since the substance group has already been very well studied as a disinfectant, Hirt is confident of obtaining the approval. After all, a replacement fixative will be needed in the future. Hirt comments: "Physicians learn the most when they work on a fixed cadaver and are able to touch the muscles and organs and thus understand three-dimensional structures and the relationship to other structures. This cannot be achieved with simulations,” says Hirt.

References

(1) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Formaldehyde, 2-Butoxyethanol, and 1-tert-butoxypropan-2-ol. Vol. 88 (2006). Ed.: International Agency for Research on Cancer (IARC), Lyon, France. https://monographs.iarc.fr/ENG/Monographs/vol88/mono88.pdf

(2) Commission Regulation (EU) 605/2014 of 5th June 2014 amending Regulation (EC) No. 1272/2008. Official Journal of the European Union No. L167 (2014), p. 36-49

(3) Statement on formaldehyde in TRGS 900. Committee on Hazardous Substances (February 2015)

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