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.... Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=5039