X-Message-Number: 5039
Date: Sun, 22 Oct 1995 23:20:55 -0700 (PDT)
From: Doug Skrecky <>
Subject: Cryonet Gulch

                         CRYONET GULCH
                     A REPLY By Doug Skrecky
            (from Canadian Cryonics News February 1994)

     High noon. Shots rang out. A body dropped. Mike Darwin lit a cigar
 while someone else dragged the body away. As he inhaled the sweet taste
 of victory yet again the local funeral director mentioned that there was
 a new kid in town making some noises about sucrose and dry ice being
 better than glycerol and liquid nitrogen. Mike smiled and said "You'd
 think they'd never learn, nothing beats the way we do things. It's either
 our way or else boot hill and we all know what the chances of reanimation
 are for those buried in boot hill, don't we? Where is this young pup?" 
 After being directed Mike moved in aiming for another notch on his twin
 45 magnums. 
     The kid was inside sipping a glass of milk and explaining why you
 don't want to use milk sugar since it was a reducing sugar when Mike, a
 giant goliath of a man burst into the bar. *1 "Where's the Sucrose KID! 
 I'm calling him out!" The kid froze, then looked around hoping everybody
 would keep quiet about his identity. No such luck. Tom Donaldson pointed
 out "the fool is over there". Mike approached and slapped the kid on the
 back, who promptly did a head slam on the glass of milk. Mike eyed the
 kid's armament, which consisted of only a slingshot in his back pocket
 and challenged "I'm the biggest and baddest cryonics man in this town. 
 You going to sit there and bleed or are you going to do something? Cus if
 you don't I'm going to throw you out of town like those Summum fools." 
 The kid nodded outside and Mike being in a good humour obliged by
 stepping outside. "Don't do it. Mike really is the most experienced
 cryonics enthusiast in town" urged Ben Best. The kid stepped outside. 
     It wasn't high noon and it wasn't a fair fight. The kid knew this and
 knew the reputation of his opponent and that there was not much of a
 chance of killing his opponent. Who ever said life had to be fair? Mike
 fired off first. "You say dry ice storage is cheaper than liquid
 nitrogen. Wrong! Dry ice is twice as expensive as liquid nitrogen." The
 kid was still standing and indicated this by replying that liquid
 nitrogen boils off a lot faster than dry ice sublimates. This same
 volatility requires vaccuum insulation to make the liquid nitrogen last. 
 Vaccuum insulation requires a very expensive containment system which
 thus far has prevented room sized containers from being built. Dry ice by
 contrast should not require vaccuum insulation and a room sized
 containment system might then be built quite inexpensively by for example
 lining the walls, floor and ceiling of a room with a foot or two of
 insulating foam. 
     However the real advantage of using sucrose and storing in dry ice
 would be the inclusion of a desiccant which would freeze-dry patients and
 render them virtually invulnerable to the vicissitutes of room
 temperature storage. The last time a cryonics company became insolvent
 this had rather unfortunate consequences for most of its charges. Perhaps
 the next time this happens the results will be different. The kid
 finished by suggesting that since Robert Ettinger had more experience
 with dry ice that he decide which of them was right on the economics of
 this issue. 
     Mike nodded and then let loose another blast. "You state that a
 vitrified solution is completely frozen below the glass transition. 
 According to a prominent organ cryopreservationist a vitrified solution
 is considered to be vitrified and not frozen." The audience boos "cheap
 shot". The kid blinks and demands to know what the practical upshot of
 all this semantics was. Both ice crystals and vitrified amorphous liquids
 are inert solids. While he admits that he is technically in the wrong,
 the kid just shrugs. 
     Mike blasts "You claim the glass transition or Tg for sucrose is -46
 C and for glycerol is below -65 C. Wrong! Neither sucrose nor glycerol
 possess unique glass transition temperatures. For a 96.4% glycerol
 solution it is -86, for 90% it is -92, for 80% it is -100 C. The kid
 swayed, he looked like he was hit! No! He was still on his feet. Mike
 scratched his head and then demanded an explanation. The kid begain by
 apologizing for not using the correct technical term which was Tg' rather
 than glass transition. The Tg or glass transition does vary depending on
 the concentration of the solute, but by definition Tg' is always the
 glass transition for the maximally freeze concentrated solution, which
 for sucrose really is -46 C. *2 The only figuare for the Tg' which the
 kid could find for glycerol was a suspect -65 C, which is why he
 specified below -65 rather -65 itself. *3 This same reference quoted the
 Tg' for sucrose as -32 C, which is wrong. The kid admitted he was
 misinformed about the Tg' for glycerol, but added that he wasn't very
 concerned since the mistake was made regarding glycerol, which he
 considered to be an inferior cryoprotectant which should not be used,
 primarily because it destabilizes frozen tissue at dry ice temperatures. 
     Mike blasts again "This Tg' is not a magic number which guarentees
 safety. Dr Greg Fahy states that to be safe storage temperature should be
 15 to 20 C below the glass transition." The kid replied that -46 minus 20
 equals -66 C. Dry ice is -78 C. So there's no problem. 
     Mike glares eye to eye with the kid. "One of us is going down and I
 ain't figuaring it to be me. A high glass transition requires a highly
 concentrated solution. The viscosity of concentrated sucrose solutions is
 so high that you couldn't perfuse the patient with it. Even if you could
 very little sucrose could penetrate into cells because of its high
 molecular weight. You're toast mister." 
     The kid looked a little singed, but replied that there is no need to
 perfuse with a concentrated sucrose solution to obtain a glass transition
 of -46 C. One could use a dilute solution and then anneal the frozen
 tissue at between -34 and -46 C. Below -34 ice crystals formed in frozen
 sucrose solutions do not melt, so they can only grow larger. As they grow
 larger the concentration of sucrose in the unfrozen phase increases till
 it reaches the maximum possible. At -35 C for example a 68% sucrose
 solution is maximally freeze concentrated after 5 hours. *2
     The fact that only a limited amount of sucrose penetrates cells may
 not be an insurmountable problem as only a little is apparently required
 to prevent freezing induced denaturation of cellular constituents. 
 Replacing glycerol with sucrose actually improves the survival of sperm
 for example. *4 Even supposing that the relative lack of penetration into
 cells does turn out to be a limiting factor there is nothing stopping one
 from opening up cellular membranes to afford a more rapid penetration. 
 Zinc ions for instance have a dramatic effect on both inhibiting DNA
 fragmentation and opening up cellular membranes permanently. *5 They also
 increase the stabilizing effect of sucrose on protein against either
 freezing or freeze-drying stresses. *6 *7 A less toxic means for
 acheiving enhanced permeability would be to use electroporation, which
 involves the use of a millisecond electric pulse to temporarily open up
 membranes for several hours so that molecules 3 times as large as sucrose
 can readily enter cells. *8 Other possibile strategies would be either
 the use of BHT or perhaps a lower perfusion temperature to destabilize
 membranes. *9
      Mike blasts "Saturated sucrose solutions readily crystalize. 
 Patients candied with sugar as you suggest may suffer from this." The kid
 mentioned that this would not be a problem at dry ice temperatures. Even
 at -35 C no sucrose crystalization was detected over a period of several
 weeks. *2
      Mike fires a parting shot "I don't like you mister. Your 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." The kid looks shocked and stares
 disbelieving at the blood, his blood dripping in the dust, then topples. 
 The funeral director ambles over, but looks disappointed. "This one is
 still alive Mike." Mike orders "Better give him to MaryLoo to patch up. I
 ain't never yet shot a man that was down. Besides maybe there's something
 useful in some of his malarkey after all." 

 *1 "The Effect of Carbohydrate Additives in the Freeze-Drying of Alkaline
 Phosphatase" 86-93 Vol.45 1993 Journal of Pharm. Pharmacol. 
 *2 "Amorphous State and Delayed Ice Formation in Sucrose Solutions" 
 553-566 Vol. 1991 International Journal of Food Science and Technology
 *3 "Beyond Water Activity: Recent Advances Based on an Alternative
 Approach to the Assessment of Food Quality and Safety" 115-360 Vol.30
 No.2-3 1991 Critical Reviews in Food Science and Nutrition
 *4 "Effects of Various Cryoprotective Agents and Membrane Stabilizing
 Compounds on Bull Sperm Membrane Integrity After Cooling and Freezing" 
 32-44 Vol.30 1993 Cryobiology
 *5 "Inhibition of Apoptosis By Zinc: A Reappraisal" 1256-11 Vol.187
 No.3 1992 Biochemical and Biophysical Research Communications
 *6 "Cryoprotection of Phosphofructokinase With Organic Solutes: 
 Characterization of Enhanced Protection in the Presence of Divalent
 Cations" 505-512 Vol.250 No.2 1986 Archives of Biochemistry and
 Biophysics
 *7 "Stabilization of Phosphofructokinase With Sugars During
 Freeze-Drying: Characterization of Enhanced Protection in the Presence of
 Divalent Cations" 109-115 Vol.923 1987 Biochimica et Biophysica Acta
 *8 "Electroporation of Mammalian Skin: A Mechanism to Enhance Transdermal
 Drug Delivery" 10504-10508 Vol.90 1993 Proceeding of the National Academy
 of Science
 *9 "Effects of Butylated Hydroxytoluene on Membrane Lipid Fluidity and
 Freeze-Thaw Survival in Mammalian Cells" 317-322 Vol.23 1986 Cryobiology
  
   ......Well, the Sucrose Kid died anyway. It is a pity, but the lack
 of membrane permeation for sucrose ultimately proved to be fatal. Perhaps
 this could be overcome in future by temporarily permeabilizing membranes
 with magnetic fields, but for the present sucrose is back in the
 kitchen. 
     The Sucrose Kid has a brother called the Sorbitol Kid and he
 figuares on doing a lot better against Big Bad Mike. Sorbitol can
 (slowly) penetrate membranes and has performed very well in cases where
 sucrose has failed. (Cryobiology 332-341 Vol.29 1992) However sorbitol
 has a dry Tg of only -2 C so room temperature storage of freeze-dried
 sorbitolized tissue is not going to be possible, but by the same token
 liquid nitrogen is not going to be required either. 
     Perhaps some day Mike and the Sorbitol Kid will have a punch-up. When
 it happens be sure to read about it in Cryonet Gulch II.... 


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