X-Message-Number: 2510
Date: 05 Jan 94 04:23:48 EST
From: Mike Darwin <>
Subject: CRYONICS
The Hard Rock Candy Mountain:
A Response To Douglas Skrecky's High Temperature Cryonics (Message:
#2497)
I was hoping that I wouldn't have to write this
response and that others would come forth to deal with
the issues Mr. Skrecky raises. Alas, enough time has
gone by that it appears this isn't going to happen,
and I have received a few calls from the
scientifically naive asking "Could this really work!."
There is also the issue that a person such as the
President of Alcor has actually asked that these
issues be addressed. Thus, they can't really be
ignored. (Yes, Thomas Donaldson did respond, but
however adroitly you do it, calling someone a fool is
not sufficient in and of itself.)
I would like to start by dealing with some specific errors
Mr. Skrecky makes and then go on to discuss broader issues
raised by his writings which I feel are important for those
in the cryonics community to be aware of, if not address.
In order to simplify my responses and reduce the risk of
misquoting Mr. Skrecky I will reprint his text and comment
on it as appropriate.
HIGH TEMPERATURE CRYONICS By Douglas Skrecky
>Long-term storage of biological materials has
traditionally been atliquid nitrogen (-196C) temperatures.
This may have been a costly mistake. It now appears that dry
ice (-78C) temperatures are all that is required.>
Mr. Skrecky doesn't tell us WHY this may have been a costly
mistake. If he speaking is about biological costs he may or
may not be right, but has yet to prove it (see my discussion
below) . If he is speaking about the economic costs (i.e.,
that storage at dry ice temperature will be cheaper) he
will have to prove that assertion more rigorously than by
just stating it. Liquid nitrogen storage is remarkably
cheap: one reason being that the cost of dry ice is roughly
twice that of liquid nitrogen.
While it is true that higher temperature refrigeration using
either mechanical refrigeration or liquid nitrogen as the
refrigerant is possible, designing and building such systems
is a formidable task and it is not clear that such systems
will be "less costly" to operate. Certainly they will NOT
be less costly to build and for various rather inflexible
reasons of physical law (see the earlier Cryonet discussion
on high temperature storage) they will be most economical
when built *large*. The estimated *marginal* costs for
storing whole body patients at CryoSpan is about $1200/yr.
That includes a large safety factor in the form of a large
heat sink (the liquid nitrogen) and the relatively high
degree of certainty on both theoretical and practical
grounds that both structure and biochemistry are conserved
at that temperature more or less indefinitely (1). This is
certainly not the case for higher temperatures of storage
and certainly not the case for any of the schemes which Mr.
Skrecky proposes.
> Deterioration of frozen tissue has been regarded as a
common feature at all temperatures above the glass
transition temperature. This varies depending on how quickly
freezing is carried out, as well as the nature of the
cryoprotectant solute. When a solution first freezes it
forms a mixture of ice crystals and a freeze-concentrated
liquid which holds a higher concentration of the solute than
the bulkunfrozen solution. If the temperature is decreased
slowly, the liquidphase will become maximally freeze
concentrated so that its viscosity rises to the maximum
possible for a given solute, irrespective of the original
concentration in the bulk solution. >
The above statement is essentially correct. This is one of
the frustrating things about Mr. Skrecky's writing/thought
is that he articulately blends complex fact with
distortion/misunderstanding so seamlessly as to make the
latter seem credible, as is illustrated by his next
sentence:
>This process, which is called annealing, raises the glass
transition to the highest temperature possible. As the
temperature further decreases to this glass transition, the
viscosity increases to the point where the liquid becomes a
glass and the solution is only then considered to be
completely frozen.>
The above statement, to quote a prominent organ
cryopreservationist specializing in vitrification of
mammalian organs is "gibberish." First of all, a vitrified
solution is not considered to be completely frozen. It is
considered to be *vitrified.* Freezing involves
organization of the molecules of the solution into highly
ordered patterns called crystals. A vitreous material is
amorphous or in other words a "solid solution." The
molecules comprising the solution maintain a relatively
disordered pattern and are not crystalline. I am not toying
with Mr. Skrecky here over some semantic quibble. This is a
serious issue and those involved in tissue cryopreservation
take care to separate the use of the word freezing from
vitrification. Further, the process of reaching the degree
of maximum crystal formation for a given mixture of agents
is not the process of annealing; particularly not when we
speak of annealing glasses or metals. A glass is often
annealed by holding at a fixed temperature or rewarmed
followed by slow cooling to allow the molecules in the glass
to reach equilibrium condition. If cooling proceeds too
rapidly past the glass transition point (i.e., the
solidification point of the liquid) the molecules do not
have time to reach their equilibrium relationship with each
other. This results in macroscopic strains in the material.
These strains may be eventually manifested in the form of
cracks or fractures during cooling or during addition of
mechanical or thermal energy. (Please note that these
observations apply to discussion of homogeneous solutions:
not brains or bodies which are composed of many different
components and present a far more complex situation.)
> The glass transition for pure water is -135C, while
that for most slowly frozen foods varies from -45 to -15.
The use of glycerol as a common cryoprotectant lowers this
transition since glycerol solutions form glasses only below
-65C. *1 The glass transition for sucrose solutions was
originally believed to be -32C, but recently a more critical
examination yielded a temperature of -46C. *2>
The last sentence as it stands is incorrect. There is no
ONE glass transition temperature for sucrose or glycerol
solutions. What one gets in reality is a range of Tg's
which are dependent upon the concentration of glycerol or
sucrose in the solution. For instance, these numbers for
glycerol were first determined by Luyet and Kroener in
their now classic paper "The temperature of the "glass
transition" in aqueous solutions of glycerol and ethylene
glycol (2). They found that for glycerol -water solutions
Tg versus concentration was as follows:
All concentrations are of glycerol in water on weight/weight
(w/w) basis):
96.4%= -86C, 90%= -92C, 80%= -100C, 70%= -106C, 60%+= -
111C, and 50%= -115C.
The nice thing about Luyet and Kroener's data is that it
more or less fits a straight line which increases our
confidence in it. As we can see from the above there is no
single Tg for glycerol water solutions. The Tg of
biological materials containing cryoprotectant is further
complicated by the presence of colloidal sugars or starches
(dextrans or HES), salts, and tissue lipids and proteins.
And it IS important to know the Tg of the material you are
proposing to store because, contrary to Mr. Skrecky's
implication that Tg is somehow some magic transition point
into biological safety, it is not. Both near and below Tg
crystal propagation and diffusion can still occur and the
system is by no means stable (in fact, changes which occur
below Tg are well known and such solutions are classified as
"metastable"). In fact almost the whole of volume 10 of
Biodynamica (Luyet's journal) deals with this issue and it
has remained a fertile topic among glass chemists during the
ensuing 25 years (3). In my conversations with organ
vitrification researcher Dr. Greg Fahy he has indicated that
the best thinking is that safe long-term storage for
biological systems will have to be pursued at 15 to 20oC
BELOW Tg for that system. As is usually the case in the
real world there is no magic number or magic solution to
complex and difficult problems.
>This advantage of sucrose over glycerol also extends to
temperatures above the glass transition as sucrose has been
found to be more effective than glycerol in inhibiting
protein denaturation in frozen tissue stored at -20C. *3
Would dry ice temperatures be sufficient to preserve tissue
indefinitely? It seems so. Low density lipoprotein treated
with sucrose, sodium chloride and EDTA and stored at -70C
showed no signs of either oxidative or proteolytic
deterioration over an 18 month period and when thawed
retained functionality similar to fresh LDL. *4>
These observations are all very nice but what do they have
to do with the cryopreservation or room temperature
preservation of human brains and bodies? (Also at what
sucrose concentration do these effects occur?) For one
thing, sucrose has a molecular weight of 342.3 making it
virtually impermeant to most mammalian cells. Another point
worth noting is that LDL is a storage and transport protein,
not an active catalyst such as an enzyme. This is rather
like comparing the effects of a preservative on an auto
engine and on an empty 55 gallon drum.
Sucrose's cellular impermeability is a well known phenomenon
and for this reason it has been and is used by organ
preservationists (including by Biopreservation) to act as an
impermeant species to prevent cell swelling during
hypothermic organ and tissue storage (i.e., storage above
0oC). As has been documented in CRYONICS the cryonics
community has been aware of sucrose's protein/membrane
stabilizing effects and for awhile sucrose was substituted
for mannitol in human perfusion solutions. This was also
done because of sucrose's superior (to mannitol) solubility
and glass forming characteristics.
However, sucrose does not penetrate mammalian cells to any
great degree and Mr. Skrecky fails to address the issue of
what happens to the intracellular milieu during his high
concentration sucrose treatment. But the problem is deeper
still. How do we deliver 80% sucrose to the patient's
tissues and cells? Has Mr. Skrecky ever made up an 80%
sucrose solution and LOOKED AT IT? More to the point has
he measured its viscosity or even looked it up in the
*Handbook of Physics and Chemistry*? How does one perfuse
a solution with the viscosity of 80% sucrose in water?
The relative viscosity of a 74%(w/w) solution of sucrose in
water (the HIGHEST concentration given the Handbook) is
1628. (Relative viscosity is the ratio of the absolute
viscosity of a solution to the absolute viscosity of water
at 20oC.). For a 76% (w/w) glycerol solution the relative
viscosity is, by contrast, 40.5. This, by the way, is a
9.88 Molar glycerol solution. The maximum Molar
concentration of glycerol which we QUESTIONABLY been able to
perfuse in dogs under optimum conditions is 7.5 (or 60 w/w
glycerol). That has a relative viscosity of 10.6! At the
conclusion of such a perfusion the mean arterial pressure
(MAP) is up around 120 mmHg!!! as contrasted with a normal
MAP of 60 mmHG. This solution is about as thick as
Hershey's chocolate syrup at room temperature or warm corn
syrup (it is thinner than honey but thicker than molasses).
It is questionable whether we are perfusing the capillaries
with this solution. For instance, even though our MAP is
120 mmHg our flow is down from 1500 cc/min. to 400-500
cc/min.
Let's assume we could perfuse a sucrose solution with a
relative viscosity of 10.6 (46% w/w). How would we get it
into the intracellular compartment?
>What would be the best cryoprotectant solution? Sodium
chloride depresses the glass transition, so replacing it
with potassium chloride might be a good idea. EDTA is an
effective antioxidant when the storage temperature is -20C,
but its value at below the glass transition remains to be
proved. *5 Dietary supplementation with vitamin E improves
the resistance of postmortem tissue to oxidation.*6 In any
case, long term storage must be in an oxygen free
environment as even glasses can oxidize. Adding egg yolk to
sucrose improves the survival of sperm by stabilizing
membranes during freezing and thawing, so this would appear
to be a desirable addition. *7>
The above is idle speculation and its relevance to intact,
nucleated mammalian cells, let alone organ preservation is
unproved and speculative. In fact, glutathione and other
antioxidants are included in human cryopreservation
solutions and in pretreatment protocols (i.e., pre treatment
protocols given to the patient prior to legal death) and
have been for years. The value of these agents has been
verified not only in the field with actual human patients
but has been verified in our laboratory using a live dog
model and in many other laboratories, both experimental and
clinical, for use in hypothermic organ preservation and
mitigation of ischemic insult. (I am ommitting references
here since a comprehensive list would comprise well over
100: I can supply them to any who REALLY want them).
> The replacement of glycerol by sucrose could do much to
reduce the costs of cryonic storage by enabling the
replacement of liquid nitrogen refrigerant with dry ice, but
why stop here? Unlike glycerol, sucrose is an effective
anhydroprotectant in addition to being a
cryoprotectant.Partially drying tissue by pumping dry gas
through the cardiovascular system could eliminate all damage
due to ice crystal formation during freezing since 80%
sucrose/20% water mixtures do not freeze, but instead
vitrify directly to a glass at -46C. With further
desiccation the glass transition is increased to
29C for 96.5% sucrose and 62C for anhydrous sucrose. *2 Thus
the replacement of glycerol by sucrose might very well
eliminate any need for refrigeration. *8>
Here Skrecky proposes to go from an unmentioned
concentration of sucrose (lets say 46% ) to some
concentration approaching 96.5% (which will yield a Tg of
29C) by pumping air through the circulatory system. Mr.
Skrecky gives us no numbers or indications as to how much of
the capillary bed he can access with air (keeping in mind he
is trying to displace a solution with a ten-fold greater
relative viscosity than that of water!). Blowing the
capillaries clear of this hyper viscous solution would be a
challenge indeed: how do you avoid opening a FEW capillaries
after which time you will get channeling of almost all you
gas flow through these vessels -- even at astronomically
high pressures. Remember the incredibly high surface
tension and viscosity of the sucrose solution compared to
air or other gases. And how long this would take, at what
temperature it will take place, AND what will be happening
to the patient's biochemistry and/or structure in the
meantime.
Mr. Skrecky also leaves unaddressed the problem of
*crystallization of the sucrose.* A 96.5% solution of
sucrose if it is not already crystalline will soon become
so, particularly if stored near its Tg. Anyone who has kept
a jar of honey (a concentrated solution of fructose and
organics) around long enough will appreciate this fact.
Indeed, anyone who has put a string into a concentrated
solution of sucrose will know the result: rock candy (large
crystals of sucrose!). Freezing patients is bad enough but
candying them (which is incidentally the process of
preserving biological matter by dehydrating it with sucrose)
and then turning them into mountains of crystalline rock
candy hardly seems a good approach to biopreservation. In
fact, this is all too close to what we are achieving right
now by our current methods of cryopreservation. However, at
least when we are through glycerolizing (dehydrating) and
crystalizing our patients we at least have the assurance
that they are biochemically and ultrastructurally stable
more or less indefinitely (due to the low storage
temperature). This is an assurance we do NOT have with Mr.
Skrecky's proposed approach.
These are the high points of the problems I see with Mr.
Skrecky's ramblings. The problem here is that Mr. Skrecky
is engaging in armchair science. This is very different
than armchair hypothesizing which is where much good science
starts (and regrettably, ends!). Looking at the literature
and coming up with ideas about what should work is fine.
It is often a GOOD first step in doing GOOD science. The
next step is to try to figure out holes or problems in your
"hypothesis" and a good way to test this out is to consult
with colleagues AFTER trying very hard to punch holes in it
yourself (this saves time for your colleagues, improves
their estimation of you, and often stops you from looking
silly or lazy).
Hugh Hixon has observed that Skrecky's speculation would be
more tolerable if it was put forth in a more balanced form
with some thought given to potential problems and defects
and a less panacea oriented method of presentation.
Finally, some more general observations about this matter:
1) A workable technique of high temperature (i.e., room
temperature or slightly below) biopreservation would be a
VERY desirable thing. AND there is much relatively simple,
inexpensive research which might be done by any truly
interested in pursing this option. Fixative perfusion of
brains follwed by plastic impregnation with follow up
electron microscopic studies at intervals of time usuing
accelerated aging techniques (such as holding at elevated
temperatures: say 60-70C) might be a good place to start.
(However, there are some caveats to these methods which must
be considered:
a) Fixatives must be able to REACH the tissues and that
means prompt treatment after legal death and maximum effort
exerted to maintain patent circulation and minimize ischemic
injury. This translates to the same high-level of initial
transport care as would be given to any patient to be
frozen. And that means the same high cost.
If you doubt the importance of this talk to any electron
microscopist about what kind of results you get in terms of
ultrastructural preservation with even MODEST amounts of
post mortem delay (and I mean like 30 - 60 minutes!).
b) The advantage to cooling is that it allows for inhibition
og biological activity independent of capillary
access/chemical diffusion. It thus can be used to treat a
wide range of patients; many of whom we know from experience
will not perfuse well and thus who will not fix well.
c) We have zero feedback about the effects of fixatives on
the ultimate recoverability of the tissues treated. We have
considerable feedback from cryopreservation techniques in
that cells, tissues and a few organs do recover from such
treatment. The effectiveness of fixation in preserving
essential biological structure (i.e., that required for
resumption of function) is theoretical. I tend to think
that the theoretical reasons for believing it adequate are
reasonable, but it is a much more poorly supported gamble in
terms of hard evidence.)
I bring this matter of high temperature storage up because I
feel it merits serious attention. However, the way Mr.
Skrecky has approached it does not constitute such.
2) While I favor freedom of inquiry, open exchange of ideas
and ease of publication, I also believe in filters. NATURE,
SCIENCE, or any responsible publication, scientific or not,
does not indiscriminately publish anything that crosses its
desk. To do so is irresponsible and puts responsible
persons in the position of having to expend their time
rebutting useless claptrap. (Something I have just been
doing). This is why peer-reviewed journals came about and
why even popular publications use a panel of experts or
responsible advisors to screen the material they publish,
particularly where it involves technical (i.e., more
objective) matters and especially when it involves matters
of life or death. For instance, the *LA Times* would
carefully check any article purporting to report a cure for
cancer; or they would present the material with cautions and
disclaimers quoting contrary opinion, etc.
I believe that CANADIAN CRYONICS NEWS and other cryonics
publications have a similar level of responsibility. To
publish a piece such as Skrecky's *first*, without any kind
of disclaimer or caution is irresponsible. Such is both bad
science and bad journalism and in my opinion cannot be
excused by an "open" editorial policy.
*My thanks to Hugh Hixon for his valuable criticisms and contributions
to this response. Thanks also to Steve Bridge for his
comments/corrections.
** Mr. Best has not only my permission, but my encouragement to print
this in CANADIAN CRYONICS NEWS.
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