Sabine Rosenberger

Sabine is a mouse midwife. She’s looking after about 20 expectant mouse moms, and the prognosis for the babies is not good. 3/4 of them will be born looking relatively normal, and no one is too concerned about these. The other 25% will be born with a severe, congenital defect of the skin and will be unable to seal up a water-tight barrier between themselves and the surrounding world. They will dry up like raisins and die of dehydration within 3 hours.

Sabine’s mouth is wide and full, with heavy lips that seem pushed forward in the early phase of giving someone a kiss. If you tease her, the lips will part showing big, healthy, white, German teeth. Her eyes are misty, gentle, and a bit sad, and without the watchful eyes of Sabine and her coworkers, no one would ever see these mice. They would be eaten by their own mothers and disappear from the litter, as if by magic. This is exactly what happened for the first 6 months as they tried to raise these special 12(R)LOX deficient mice. There just didn’t seem to be any. Then one night, by sheer random chance, she stumbled on a full litter minutes after they were born, and she saw the tiny, shriveling raisin babies.

12(R)LOX-/- knockout mouse pup

To understand why Sabine goes without sleep night after night, watching so that she can be a part of the brief, 3-hour lifetime of some tiny, sick, naked mice, it helps if you like chemistry. The popular journalist  @edyong209  likes to joke that he is a human-shaped bag full of microbes, but that’s not really correct. He’s a bag of salt water, with a long, smelly tunnel punched through it from one side to the other. Most of the microbes live in the tunnel, not in the water bag. The fact that all that salt water doesn’t just leak out of the bag is because of the remarkable, saran-wrap-tight seal that our dermal epithelial cells create as they finish their life cycle and cement themselves together just before they die. Mice and humans exist inside a smooth, elastic, water-impermeable skin with an outer layer that is made of senescent keratinocyte bricks mortared together with a cross-linked lipid and protein glue. For a cartoon of this, see Figure 1.

Figure 1

Each little zipper link connecting adjacent keratinocytes is composed of an omega-hydroxylated very long chain ceramide (30 to 36 carbons in length) covalently attached to glutamate. This cell-to-cell zipper only forms if the essential fatty acid linoleate is first attached to the end of the ceramide and then oxidized in-situ by the 12(R)LOX enzyme.

For almost my entire adult life, I have studied the catalytic biochemical reactions between oxygen atoms and the polyunsaturated fatty acids that reside in cell membranes. One of those reactions is catalyzed by the COX enzymes, and I wrote about it here. Another is catalyzed by the 12(S) LOX, and I wrote about it here. The making of skin glue is catalyzed by its close relative, 12(R)LOX.

It does not seem intuitively smart to set fire to the oils and lipids that make up the membranes of your skin cells, but as long as you do it in a slow, controlled way, adding exactly 2 molecules of dioxygen to each 18 and 20 carbon fatty acid, it’s not only a good idea, it’s essential to survival.

Polyunsaturated fatty acids are not formally called “vitamins” but they should be. Vitamins were named for the letters A through E, but then biochemistry seems to have gotten drunk or tired and lost its consistency. The use of Vitamin F for Fatty Acid was proposed, but because it was complicated, it fell into disuse. Some fatty acids cannot be made by humans, must be obtained in the diet, and if absent from the diet, cause a deficiency disease state that was first observed by George and Mildred Burr in 1930 in rats (PDF). Since doctors receive almost no training in nutrition, they rediscovered this disease in humans when they tried to feed patients entirely by vein in the 1970s (PDF). Very sick adults and neonates born with digestive system problems were fed intravenously, and the early TPN (Total Parenteral Nutrition) formulas contained no essential fats. Scaly red skin, kidney, neurologic, and immune system problems developed in these neonates, faithfully replicating the forty-year-old rat data. Today one cannot watch 30 minutes of television without being pitched several different brands of omega-3 fats, but unless one trusts an adman with science outreach, a refresher is always helpful.

Because the 12(R)LOX enzyme is missing from the little knockout mice that Sabine is caring for, the essential linoleate substrate waiting in their skin cells fails to react properly with oxygen. That is, they have their Vitamin F1, but they can’t metabolize it correctly. With no oxygenated epoxides present in the skin ceramides, no glue forms between the dead keratinocyte bricks. They stack loosely on each other, and water vapor escapes all around them. Essential fatty acid deficiency in rats and humans also includes notably dry, red, scaly skin.

There are humans born with the same genetic abnormality as these mice. That is, they also have a non-functional, null mutation of the 12(R)LOX enzyme and likewise cannot make peroxides and epoxides of linoleate. But human babies are quite a bit larger than mice, so the process of drying out takes much longer. Their parents care for them and add lotions to their skin instead of cannibalizing them, so these babies survive. Still, they have a nasty, scaly skin that reminded pathologists of carp scales, so their disease was named Autosomal Recessive Congenital Ichthyosis (ARCI). Any defect in this pathway, from the synthesis of the very long omega-hydroxyl ceramides to the final attachment to proteins by trans-glutamase, will manifest in some form of dehydration and scaly skin.

Collodian baby born with a mutation in the 12(R)LOX/eLOX-3 pathway

In theory, one could make a chemical replacement compound for the oxidized linoleate ceramides, and these people could rub it into their skin and become more like everyone else. In practice—this has been quite hard.

When the baby mice are born, researchers quickly euthanize them and remove as much of their skin as possible. Consider what skinning a neonatal mouse could be like, and you’ll realize that’s not very much skin. The cells are suspended in medium and grown in a special 3-D culture system that lets them orient in a natural plate-like manner and try to form a skin barrier, although a very leaky one. It does not matter that it’s leaky, because the cells are surrounded by liquid culture medium. Then a compound can be applied to them, and the barrier can be tested a few days later to see if it still leaks.

The lab in Heidelberg where these experiments are conducted estimates their overhead costs to test each new experimental fatty acid I supply them at about 50,000 Euros. It takes 12 months just to get permission from the government to mate the parent mice. Early in 2015, we made it all the way through this cycle: Ask for permission and fill out numerous forms (wait, wait, wait), mate, gestate, parturate, and euthanize in Germany. Meanwhile a single hopeful new compound gets prepared in America by our chemist Andrei, who came to us from Kiev, Ukraine. The compound we made worked… a little bit. The data look “promising.” Now we can make a slight change, and maybe this fall, try a new fatty acid. Two new compounds per year can still result in an important medical advance. But only if one is very, very patient, like Sabine.

The author with Dr. Peter Krieg of DKFZ in Heldelberg

There are hundreds of naturally occurring fatty acids, but they can be lumped in three main groups. The first, including Butyric (from butter) Palmitic (from palm oil) and Oleic (from olive oil) are non-essential, and can be built in 2-carbon steps by humans if needed. The second group is the omega-6 polyunsaturates. Of these, Linoleic is required in substantial amounts because of its structural role in the skin. Arachidonic is also essential, but less important because it can be made from Linoleic, and is needed in much smaller amounts to make potent lipid signalling molecules like Prostaglandins. The third group, the omega-3 fatty acids, are the ones most often pitched as supplements. Alpha-linolenic from flax seed oil is the poor man’s omega-3. It is cheap, can only be converted by the body into the crucial DHA omega-3 inefficiently, and is mostly burned off for energy. EPA has no specific role of its own, but it is almost always present in fish oils in amounts comparable to DHA and the two are readily interconverted. DHA has crucial structural roles like Linoleate, but the case for DHA being the parent of a potent lipid signalling cascade is much less firm than is the case for arachidonic acid. It is important to note that the omega-6 and omega-3 families cannot substitute for each other. A few grams of vegetable oil and a few grams of fish oil on a weekly basis will prevent EFA deficiency. Perhaps, on an appropriate day, a serving of fish and chips.