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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