14. September 2008 · Comments Off on D(+)-Lactose and other sugars in organ preservation and cryonics · Categories: Cryonics, Science · Tags: , , , , , ,

A PDF file of this document is available with images and structural visualization of various sugars.

D(+) lactose monohydrate is the principal sugar in mammalian milks. The monohydrate part is easiest to explain; it simply means that the lactose molecule has one water molecule attached to it. This is important because some chemicals can have a lot of water molecules attached to them. For instance, you can have magnesium chloride with two attached water molecules (dihydrate) or six attached water molecules (pentahydrate). This becomes very important when you are weighing out a chemical and you need the chemical to be present in the correct amount. You’ll understand how important this is if you consider that someone proposes to sell you a kilo of some very valuable chemical (say 100 times more valuable than gold per milligram). There is going to be a considerable difference in the amount (by weight and usually by volume) of the actual active chemical you get per milligram or gram (weight) depending upon how hydrated it is (how many water molecules it has attached. The molecular weight (molecular mass) of magnesium chloride is 203.30 and the molecular weight (MW) of water is 18.01. Now, if you have 6 water molecules for each magnesium chloride molecule you have a total mass of water of  (18.01 x 6) = 108.06. That means if you have the pentahydrate salt of magnesium chloride you must add the weight of the 6 water molecules to the MW of magnesium chloride: 203.30 + 108.06 = 311.36. So, if someone is selling you a gram of magnesium chloride pentahydrate at the same price you can buy magnesium chloride anhydrous (no water) you are getting cheated because you are paying the same price for a gram of product that is 1/3rd water!

In biology and chemistry the same principle applies because if you need a certain amount of a chemical for critical reasons, say to maintain normal cell function or inhibit cell swelling in hypothermia, then you must account for any water molecules that may be attached to the chemical. In the case of magnesium chloride pentahydrate versus anhydrous magnesium chloride you are going to have to weigh out about 1/3rd more of the powder of the pentahydrate salt in order to get the same amount of magnesium chloride present in one gram of the anhydrous salt.

Why have pentahydrate, monohydrates, dihydrates and so on of chemicals? The answer is that some chemicals are almost impossible to handle in room air without rapidly absorbing water. Some chemicals will absorb just so much water and no more and thus are very stable under conditions of normal use, so they are supplied in this form. Some chemicals, especially organic chemicals, are virtually impossible to economically produce without one or more attached water molecules. Magnesium chloride is a really good example because it is intensely hygroscopic; it will literally pull water out of the air right before your eyes. So, not only is anhydrous magnesium chloride more expensive than the pentahydrate, it is virtually impossible to handle. If you try to weigh it out it will literally be grabbing water from the ambient air so fast that you can’t tare it on the scale. In seconds you will see tiny droplets of water on the weighing boat or paper where the magnesium chloride has literally pulled so much water out of the air it is fully dissolved in a tiny droplet of water! Calcium chloride is just about as bad, so, you’ll notice that we don’t even bother trying to weigh these chemicals out as dry powders, but rather buy them as pharmaceutical products for injection because they are already dissolved in solution in very precise concentrations. Thus, it is much simpler to draw up the correct volume of these salts dissolved in solution to add the desired amount of these two chemicals to perfusate. It is possible to weigh them, but you have to be quick and it helps if you live the desert where the humidity is very low.

Now we come to the D(+) part which is much harder to explain. In the early part of the 19th Century the French physicist Dominique F.J. Arago noticed that when he passed polarized light through quartz crystals the light could be rotated either to the left or right depending upon the individual crystal. Shortly thereafter the brilliant physicist and mathematician Jean Baptise Biot (the Biot number, a dimensionless number used in unsteady-state (or transient) heat transfer calculations, is named after him) also observed this same effect in liquids and gases of organic substances such as turpentine and some other petroleum products. About 10 years later the English astronomer Sir Joun F.W. Herschel discovered that different crystal forms of quartz rotated the linear polarization in different directions. Simple polarimeters have been used since this time to measure the concentrations of monosaccharide sugars, such as glucose, in solution. In fact, one name for glucose, -dextrose-, is so named because it causes linearly polarized light to rotate to the right or “dexter” side. Similarly, levulose, more commonly known as fructose (fruit sugar) causes the plane of polarization to rotate to the left. Fructose is even more strongly levorotatory than glucose is dextrorotatory. Invert sugar which is formed by adding fructose to a solution of glucose, gets its name from the fact that subsequent structural conversion causes the direction of rotation to “invert” from right to left.

The reason for this behaviour of these seemingly identical substances was not understood until the mid-19th Century when Pasteur was working on the problem of why wine was souring as opposed to fermenting into an alcohol solution. The culprit was yeast that metabolized the fructose in the grape juice to tartaric acid. A solution of tartaric acid derived from living things (the wine lees yeasts) rotated the plane of polarization of light passing through it, whereas chemically synthesized tartaric acid prepared by non-organic means in the laboratory did not have this effect. This was puzzling because both the synthetic and the biologically derived tartaric acid undergo the same chemical reactions and are identical in their elemental (atomic) composition. Pasteur noticed that the crystals came in two asymmetric forms that were mirror images of one another. He meticulously sorted the crystals by hand and then dissolved each of the two forms of crystals in water; solutions of one form rotated polarized light clockwise, while the other form rotated light counter-clockwise. An equal mix of the two had no polarizing effect on light. Pasteur deduced the molecule tartaric acid molecule was asymmetric and could exist in two different forms that resemble one another; as would left- and right-hand gloves, and that the organic form of the compound consisted purely of the one type.

This phenomenon is referred to as isomerism and occurs when two molecules have the same molecular formula (atomic composition) yet have different structures and therefore different chemical and physical properties. There are many different kinds of isomers. The two major divisions of isomers are the geometric and the structural. Structural isomers are isomers that have the same number of atoms but different arrangement of atoms. One structural isomer of glucose is fructose. Geometric isomers are identical in arrangement of covalent bonds but are different in the order that the groups are arranged.

A major category is stereoisomers which are two isomers that have the number of atoms in the same order. A stereoisomer of glucose is galactose. In the Fischer projection all of the atoms are the same except for one rotated group. There are two categories of stereoisomers, enantiomers and diastereomers. Enantiomers are two isomers that are mirror images of each other when looked at in 3D while diastereomers are not. Galactose is just one of many diastereomers of glucose. To find out the possible number of stereoisomer forms a monosaccharide can have, you can use the formula 2x where x is the number of chiral carbons the molecule has. Molecular chirality occurs when a sugar has a carbon with four different groups attached to it. Any carbon with a double bond on it is never chiral nor are the end carbons. Because glucose has four chiral carbons there are 24 different stereoisomers; which means that there are sixteen different stereoisomers for glucose.

Two of the main divisions of glucose’s many forms are l-glucose and d-glucose. These two are enantiomers which are determined by whether the two molecules are symmetrical at the last chiral carbon. When the hydroxyl group is on the last chiral carbon on the right it is considered d-glucose  and when it is on the left it is classified as l-glucose. The “d” means that the glucose rotates polarized light to the right (dextrorotatory) and the “l” stands for  levorotary (rotates polarized light to the left). These refer to how a plane of light rotates as it passes through a solution of it. First light is passed through a polarizing filter, then a polarimeter containing a solution made with the molecule. When a d-solution is in the polarimeter it will cause the light to turn to the right or at positive angle, while an l-solution will cause the light to turn to the left or a negative angle. Both d-glucose and l-glucose exist naturally but d-glucose, also called dextrose, is the most abundant sugar on the planet.

The practical biological and chemical implications of these isomeric structural differences is profound. D-glucose (dextrose) is the principal sugar used by the body to generate energy. By contrast, l-glucose cannot be significantly metabolized and an animal or human would starve to death if this was the only carbohydrate available in its diet and no other sources of energy (fats or proteins) were available. L-glucose looks, tastes and has the same mouth feel as d-glucose and there has been considerable interest in producing it in large quantities as an artificial sweetener.  Unfortunately, the synthetic pathway to produce l-glucose, and more importantly, the separation of the d- and l-glucose isomers after synthesis is currently prohibitively expensive.

What does all this have to with cryonics and organ preservation? Under normal metabolic conditions the cells of the body produce chemical energy in the form of ATP and about 1/3rd of this energy is used to pump ions into and out of the cells. This is necessary because the most common salts (ions) are very small and can easily pass through the cell membranes. Two straightforward examples are very much on-point. Cells need high concentrations of the potassium ion inside them to be able to function properly including carrying out some vital chemical reactions and doing things like contracting in the case of muscles or transmitting signals in the case of nerve cells. Conversely, cells must not have too much sodium in them or they  become swollen (edematous) and while this can ultimately rupture or lyse the cell, long before this happens cell swelling disrupts the meshwork of supports that maintain the cell’s shape and probably serve as scaffolding for various enzymes to be anchored on and to facilitate efficient chemical processing (metabolism). Unfortunately, sodium has a net negative charge and the protein inside cells has a net positive charge. Thus, sodium will flow into cells and carry water with it resulting in cellular edema. This process is prevented by active pumping of sodium out of the cell. Similarly, calcium is extremely toxic to cellular mitochondria in high concentrations and calcium is also used as a critical signalling molecule inside cells. Thus, the calcium concentration outside cells is typically 10,000 times higher than that present inside cells. Again, this difference in concentration is maintained largely by active pumping which requires energy expenditure and on-going metabolism.

So, sodium gets pumped out and potassium gets pumped in and this process is linked and carried out by the same molecular machine; the sodium-potassium pump. Of course, all of this presumes that there is both available energy in the form of ATP and that the cellular pumping machinery can use that energy. There are a number of things that can interrupt ion pumping. There can be a lack of energy due to starvation, hypoxia or ischemia, and there can exist situations where the energy is available but cannot be used. Some chemicals poison enzymes critical to ion pumping; a classic example is tetrodotoxin which comes from blowfish and which poisons sodium pumping. The other condition where adequate energy (ATP) can exist but cannot be used is deep hypothermia. Non-hibernating animals have enzymes that shut down or become inactive when the temperature is reduced well below that of normal body temperature. In humans (and most non-hibernating mammals) the  enzyme responsible for pumping sodium out of cells and potassium into them, sodium-potassium-ATPase, is largely inhibited at 10oC and is virtually shut down at few degrees above 0 oC.

Cell swelling in brain cells occurs with incredible rapidity after interruption of blood flow in ischemia (cardiac arrest). While cell swelling is not the only, or even primary, cause of injury in cerebral ischemia, it is a major player in causing injury in cold ischemia; conditions which obviously obtain in organ preservation and ultra-profound hypothermia in cryonics patients. The way this edema is prevented in organ preservation is to replace almost all of the small cell membrane permeable ions with big molecules that cannot pass through the cell membrane and which are osmotically active; in others words can hold water outside of the cell.  The first solution to do this with any success was Collin’s Solution invented by Geoff Collins. It used comparatively large phosphate salts to keep water outside of the cells and prevent cellular edema. However, phosphates do leak across the cell membrane and they are incompatible with DMSO and also precipitate as crystals when solutions are cooled to low temperatures or frozen.

Thus, the organ preservationists turned to sugars and sugar alcohols as molecules to serve as an osmotic agent and prevent cell swelling. Sugars are comparatively large molecules and some are very large. They do not typically pass through cell membranes rapidly, if at all. Some of the first sugars tried were glucose and sucrose and the sugar-alcohol mannitol. Neither glucose nor sucrose worked well. Glucose leaks across cell membranes fairly rapidly and has facilitated transport in the brain. Sucrose makes quite viscous solutions in the necessary concentrations (~180 mM) and for unknown reasons is toxic to the kidney tubule cells. Mannitol was much more successful in the laboratory but never made it into clinical organ preservation solutions.

In the 1980s, a biochemist named Jim Southard and a transplant surgeon named Folkert Belzer began to systematically study molecules to inhibit cold ischemic swelling, as well as other molecules to help conserve ATP, inhibit free radical damage, and otherwise address the derangements that occur under deep hypothermic conditions. They identified two sugars as particularly effective in inhibiting cold ischemic cellular edema, raffinose and lactobionate. They combined these two sugars along with other ingredients to create the first and still most successful “universal” organ preservation solution, UW-Solution, or as it is commercially marketed, Viaspan.

Unfortunately, ViaSpan does not work for the brain. We tried it extensively in the early 1990s and got serious cerebral edema followed by convulsions and death in dogs that had been perfused with ViaSpan for as little as two hours! By contrast, we could recover dogs perfused with MHP (a mannitol based perfusate) after 5-hours of bloodless perfusion with the solution at  5oC with no neurological or other problems; most of the dogs were placed with cryonics members and lived out the rest of their lives normally.

Recently, 21st Century Medicine has been systematically investigating hypothermic organ preservation and they have made a number of stunning breakthroughs. One thing which was long overdue to be done was to systematically screen various molecules for their cell swelling inhibiting effects. They found that one sugar in particular was highly effective, D(+)-lactose. Only the d-isomer worked well.

Why some sugars work and others do not, or actually cause harm, is a mystery. The molecular weight is certainly a factor, but different sugars with nearly identical molecular weights may perform totally differently. Also, the isomer of the sugar appears critical in some cases, as is the situation with lactose.  21st Century Medicine has patented an organ preservation based on D(+)-lactose and is in Phase II clinical trials for this solution, which they call TranSend. They are currently getting 72-hour simple flush and store on ice preservation of kidneys (rabbit and dog), pancreases and livers (dogs) and are getting similar results in the human clinical trials. They have achieved 48-hour heart preservation using a derivative of this solution which combines periods of trickle-flow cold perfusion with brief intervals of modest warming to ~15 oC.

31. August 2008 · Comments Off on Structure-function analysis of neuroprotectants · Categories: Cryonics, Neuroscience, Science · Tags: , , , ,

In “The chemistry of neuroprotection”, the author argues convincingly that there could be great benefit from a systematic and rigorously scientific study of the physical chemistry of putative neuroprotectants vis-à-vis their pharmacological effect. However, the first example used of the earliest thinking in this direction (which comes, not surprisingly via V. A. Negovskii, the father of resuscitation (1) medicine) is instructive as to some of the potential barriers standing in the way of this approach.

“It is not surprising that all the agents which are effective in shock carry a negative charge. This applies both to heparin, which possesses a very strong negative charge, and to hypertonic glucose solution. The same may be said about a substance now in wide use – dextran – which has small, negatively charged molecules, and also about the glucocorticoids 21, 17, and 11, which also have a negative charge.” – Professor Laborit in: Acute problems in resuscitation and hypothermia; proceedings of a symposium on the application of deep hypothermia in terminal states, September 15-19, 1964. Edited by V. A. Negovskii.

In the intervening decades since Laborit wrote the words quoted above, supraphysiologic (high) steroids have not only failed to demonstrate benefit in cerebral resuscitation and shock, they have been found to be actively harmful in every well designed RCT undertaken to test their utility (a). This also extends to their lack of utility in trauma, spinal cord injury and sepsis. Similarly, the utility of heparin in treating the encephalopathy of the post-resuscitation syndrome, or improving survival after cardiac arrest has recently been called into question. Glucose, hypertonic or otherwise, was long ago demonstrated to markedly increase neurological injury if given immediately after reperfusion following cardiac arrest, and elevated blood levels of glucose, both pre- and post cardiac arrest have a strong negative correlation with both survival and neurological outcome.

Determining the seriously harmful effects of steroid administration in critical illness took decades. Despite the compelling evidence for their injurious effects, administration of large, supraphysiologic doses of steroids is still a practice both used and defended by some clinicians (albeit not ones who rely on evidence based criteria) and the use of glucose in shock, trauma and cardiac arrest took a nearly comparable period of time to discredit. These two examples are noteworthy because they comprised mainstays of therapy for most kinds of neuroinjury for decades, and they had compelling theoretical appeal, as well as many positive small clinical and animal research studies. Indeed, the debate continues to this day with controversy centred mostly on the use of low or “physiological replacement” doses of steroids in critical illness. As the eminent pulmonologist and intensivist Neil Macintyre observed in 2005, “Patients die, but steroids never do.”  This raises the twin problems of bad research (i.e., junk science) and statistically under powered or otherwise flawed studies. Combined, it has been estimated that these two types of defective studies comprise the bulk of published peer-reviewed scientific work.

High dose corticosteroid therapy for neuroinjury offers another complication in determining the therapeutic efficacy of any drug that merits consideration as a neuroprotectant (new or old). While there is no doubt that high-dose corticosteroids are ineffective and deleterious in the clinical setting, there is also little doubt that these agents are neuroprotective in the laboratory setting under certain conditions and for discrete subpopulations of neurons. The reasons for the failure of translational research in the case of corticosteroids are complex, but are mostly attributable to crucial differences between the laboratory and the real world of clinical medicine. In the case of corticosteroids these differences are most significantly:

a.    Delay from time of insult to time of treatment; in the laboratory the timing of interventions is uniform and is typically much shorter than is the case in the clinic where delays in both presentation and treatment are both long and highly variable.
b.    Heterogeneity of injury in humans compared to animals; animal models of neuroinjury are highly standardized (location, extent, mechanics) whereas human patients present with diverse injuries inflicted in many complex and often poorly understood ways.
c.    Species differences; not only are there large genetic differences between humans and rodents in general, there are dramatic differences in the native ability of rodents to both resist and overcome infection in comparison to humans.
d.    Demographics and comorbidities: laboratory animals are comparatively very uniform genetically, are typically young and healthy and of the same age, do not have comorbid conditions such as hypertension, diabetes, atherosclerosis, obesity or the diminished physiological capacity and repair and regenerative capacity increasingly present in humans over the age of 25.
e.    Rodents aren’t people and do not interact with investigators in ways that facilitate straightforward determination of an adverse affect such loss of short term memory, or other cognitive deficits. It is now understood that the corticosteroids are toxic to the neurons of the hippocampus in both rodents and men. However, injury from this adverse effect is not only more evident in men than in mice (or rats for that matter), it is only men who are capable of complaining about it.

It is notable that all of these effects, with the possible exception of increased resistance to steroid-induced immunosuppression-mediated infection, obtain in the case of other translational models of drug development. The conclusion that corticosteroids are very likely neuroprotective in humans (in terms of the direct pharmacological effect on selected subpopulations of neurons in injured central nervous tissues under ideal conditions) is highly likely. However, the confounding realities of the clinic and the genetic differences between men and rodents (the animals almost exclusively used in this type of research) mask this effect. This poses yet another serious challenge to investigators seeking to establish common moieties in prospective neuroprotective molecules.

Clinical trials of putative neuroprotective substances have been overwhelmingly negative. This has been the outcome despite often stellar results achieved in animal models; often in diverse species in studies conducted by multiple investigators in different institutions and sometimes in different countries; none of whom have any obvious relationship, let alone one that might raise the specter of conflict of interest. In the last 6 years alone, over 1000 experimental papers and over 400 clinical articles have appeared on this subject. What this suggests is that the same deficiencies seen in studies reported upon in rest of the peer-reviewed biomedical literature also apply to studies of pharmacological intervention in neuroprotection. An inevitable conclusion is that until the signal to noise ratio improves, attempts to draw general conclusions about  the shared, essential properties of neuroprotective molecules will be difficult at best, and unreliable or misleading at worst.

Perhaps a good place to start this kind of analysis is in an area where the molecular structure of the agent(s) is extraordinarily simple and the animal and clinical data are both robust and show good to fair agreement. Hypertonic sodium chloride solutions have demonstrated efficacy in providing both systemic (splanchnic) and cerebral protection in a broad class insults including hemorrhagic/hypovolemic shock, closed head injury and less robustly in stroke and global cerebral ischemia. Interestingly, other cation salts of chloride given at comparably high tonicity do not have this effect. Furthermore, animal as well as small human clinical studies have demonstrated isochloremic hypertonic solutions to be as effective as hypertonic sodium chloride at restoring microcirculatory flow and reversing metabolic acidosis in haemorrhagic shock without the potentially troublesome side-effect of raising the mean arterial pressure to levels where re-bleeding may occur in trauma or subarachnoid haemorrhage.  A relative lack of effectiveness of the chloride salt of magnesium compared to the sulfate salt of this ion has also been noted. Understanding the mechanics of these paradoxes would seem to be a worthwhile and comparatively straightforward place to begin such structure-activity relationship analyses.

17β-Estradiol

Cerebroprotective drugs not infrequently possess a multiplicity of pharmacological effects that are known to be neuroprotective but that may be accomplished by very different and even indirect means in terms of their structure-function relationship. Some cerebroprotective molecules, such as the female hormone 17β-estradiol and the mixed estrogen antagonist-agonist tamoxifen share common physiochemical properties such as free radical scavenging, N-methyl-d-aspartate (NMDA) receptor inhibition, and modulation of volume regulated anion channels (VRAC); which play a role in ischemia-induced release of excitatory amino acids. There is considerable evidence that some of 17β-estradiol’s neuroprotective effect is via signal transduction as well as its neurotrophic effects, even at doses below those necessary for its direct effects on reactive oxygen species production and its NMDA receptor inhibiting effects. While the structure of the molecules shares some important features, they are also structurally very different and the signal transduction and neurohormonal effects are almost certainly very different. Thus, these molecules also present a fascinating opportunity to probe structure-function relationships in neuropharmacology.

Tamoxifen

Finally, an admission, or perhaps a confession is order in ending this discussion. This author has been responsible for the application of at least one putative neuroprotective drug to cryopatients which ultimately proved ineffective in human clinical trials when administered during and after cardiopulmonary resuscitation (CPR). This drug, nimodipine, performed well in animal trials, but failed to show benefit in human trials, possibly as a result of its hypotension-inducing effect. Adequate mean arterial pressure (MAP) following resuscitation from cardiac arrest is essential to survival and a post arrest bout of hypertension has been demonstrated to provide substantial cerebral rescue in animal models of global cerebral ischemia. Reduction of MAP in cryopatients is a serious concern because achieving adequate perfusion pressure is problematic under the best of conditions. It is also worth noting that cryopatients have been given a variety of other ineffective neuroprotective drugs over the past 30 years, including the opiate agonist naloxone, the corticosteroid methylprednisolone and the iron chelating drug desferroxamine.

While these drugs, with the possible exception of nimodipine, are not likely to have been injurious (except perhaps to the pocketbook), their use raises important questions about when and how promising animal research should be translated to the setting of clinical cryonics. Unique among all other populations of human and animal patients, cryopatients have the opportunity to be treated with neuroprotective drugs that show great promise, absent the long delays of regulatory vetting, and independent of the economic pressure experienced by pharmaceutical companies to not only market drugs that are effective, but to market ones that are also profitable. The question thus becomes what criteria do we use in applying these drugs absent the extensive pre- and post marketing evaluation that obtains with approved ethical drugs? In essence the question we must ask and answer is “can we do better, much better in fact, than our colleagues in conventional critical care medicine?

Michael G.  Darwin, Independent Critical Care Consultant

Click here for this entry with references in PDF format.

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(1) Resuscitation medicine is properly termed reanimatology, and is so-called in the non-English speaking world

(a) The one condition in which there is unequivocal benefit to supraphysiologic administration of steroids is meningococcal meningitis with substantial evidence also supporting a similar degree of efficacy in Typhoid  and Pneumocystis carinii pneumonia.