“Put him back on ice…”

— Evil Hydra scientist Arnim Zola speaking about Bucky Barnes in Captain America: The Winter Soldier

There’s a great convergence of activities occurring right now. The Major League Baseball season is just starting up and the sporting lens is squarely on America’s Pastime. At the same time we have Captain America: The Winter Soldier the next movie installment of America’s superhero (or at least a superhero with “America” right in the name), the first of the big budget releases coming out this year from Marvel Studios.

Captain America was created by the legendary duo of Joe Simon and Jack Kirby and debuted in Captain America Comics #1 in March of 1941. He has an edge on those other grandfathers of comic book superheroes, Superman and Batman, who debuted NOT in their own books but instead in Action Comics (Supes in 1938) and Detective Comics (Bats in 1939).

Like all comic book superheroes, Cap’s origin story has been tweaked, revised, and re-written numerous times over the decades. Captain America has had a long career, but he has no supernatural powers. Cap’s Marvel Studios backstory, seen in “Captain America—The First Avenger” and the basis for the character in “The Avengers” and “Captain America 2”, is heavily influenced by the plotlines found in the Marvel Universe that holds the “Ultimates.”

In the “Ultimates” universe, the U.S. Military used a pharmacological cocktail found in Dr. Abraham Erskine’s “super-soldier serum” and “vita-ray” treatment to transform the slightly-built soldier (or actually soldier reject) Steve Rogers into the “perfect" specimen of human development and conditioning. Cap also had a wingman in the form of Bucky Barnes who was thought to have perished back in World War II.

In addition to World War II capers, Captain America and Bucky Barnes share something else in common—they have both been frozen. Cap was frozen after the plane crash that ended his first movie in 2011 and thawed out and back to active duty in “The Avengers” (2012). Bucky Barnes (aka The Winter Soldier in Ed Brubaker’s classic comic book run and in this movie) has been frozen and thawed many times over the years.

It’s this freezing and then thawing thing that’s the science focus of this blog post. That’s because in order for Steve Rogers and Bucky Barnes to survive the deep freeze they’d need to share something in common with the wood frog, Rana sylvatica.

The wood frog is a species with a huge range spanning from southern Ohio to the Alaskan interior. The wood frog can survive complete freezing of more than 60 percent of its body water and temperatures of -18 degrees Celsius in samples taken from Fairbanks, Alaska. The basic mechanism of cryoprotection in wood frogs is related to the colligative properties of body fluids, particularly a reduction in the absolute freezing point of total body water.

Cryoprotection comes about by increasing the concentrations of urea (the main nitrogen-sink found in your urine) and glucose (the simple carbohydrate found in your blood) which leads to less ice formation during exposure to sub-zero temperatures, as well as the shunting of water and thus ice formation to the lymphatic system. This is pretty critical because it reduces damage throughout the frog’s body by maintaining integrity of cellular membranes.

Jon Costanzo and colleagues at Miami University in Oxford, Ohio were interested in how the wood frog could survive sub-zero temperatures as low as -16 degrees C and even being completely frozen for two months at -4 degrees C. These animals could be thawed again at 4 degrees C and regain function. While the molecules are simple, the process of cyroprotection in a widely distributed species like the wood frog has been more mysterious.

Costanzo and colleagues found that in comparison to their Ohioan cousins Alaskan wood frogs have a much larger capacity for producing glucose, can survive with much lower glucose levels when thawed, and concentrate the cryoprotectants urea and glucose at much higher levels in the brain. This means that within the same species unique evolutionary adaptive mechanisms are found within the wood frog to produce necessary adaptations across its geographic range. In other words, it depends where the frogs live. The same group of researchers later showed that the amount of cholesterol found in the “phospholipid” cell membrane is also much lower in Alaskan frogs.

Which brings us back to Captain America: The Winter Soldier. We now know that in order to survive prolonged periods of freezing, it would’ve been necessary for something more than the super soldier serum. An additional biochemical cocktail would’ve been needed to trigger some kind of adaptive process involving glucose levels and urea handling. Which would have been rather tricky.

We also know now that the process wouldn’t necessarily have been restricted to Steve Rogers. Bucky Barnes could also have had the same adaptation. If more than one wood frog can do it, so can more than one World War II comic book patriot.