Crosslinkage Theory of Aging: Part IV AGEs and Crosslinkages – New Respect for Crosslinkage Theory
By Ward Dean, MD
Introduction and review: The Cross-linkage Theory of Aging was first proposed by Dr. Johan Bjorksten in 1941. Bjorksten believed that aging was caused by inter- and intramolecular crosslinks in proteins, nucleic acids, and other vital macromolecules that caused them to gradually stiffen and lose their function. Bjorksten initially searched for enzymes capable of dissolving damaging crosslinks. But as he grew older he realized that he didn’t have enough years of life left ahead of him to allow for the identification and isolation of these enzymes. Consequently, he shifted his line of research to a more immediately solvable approach—using chelating agents to remove toxic heavy metals (especially, aluminum)—that were known to be one cause of crosslinking. He hoped that by eliminating the crosslink-promoting tri-valent (three points of attachment) aluminum atoms (which he believed displaced di-valent [two points of attachment] calcium atoms, he would reduce one of the major sources of crosslinking, and thereby buy enough time to solve the rest of the crosslinkage problem. This has been explained in greater detail in the first three parts of this series. Bjorksten ended his active research career in 1991 with one last publication that summarized his progress up to that point. Ironically, at about the time Bjorksten was retiring from his quest to unravel the crosslinkage problem, other scientists were picking up the baton—although they approached the problem from a slightly different direction.
Advanced Glycation End Products of Aging (AGEs)
A characteristic of all long-lived proteins in the body is that as they age, they turn brown and become fluorescent (under UV light), become more cross-linked, less soluble, less elastic, and less digestible by enzymes. In 1965, Dr. H.B. Bensusan first proposed that it was a process known as the Maillard reaction that caused these changes. The Maillard reaction is named for the noted French scientist, Louis Camille Maillard (1912), who described the non-enzymatic chemical reactions between proteins and carbohydrates that cause cooked foods to turn brown. This time-honored bit of kitchen chemistry has been used by cooks for centuries to enhance flavor and transform plain foods into delicacies by adding flavor and color to recipes. In 1985, Monnier, Kohn and Cerami provided further details of the role of the Maillard reaction as a major source of the age-dependent increase in browning, fluorescence and crosslinking of collagen and other tissues. (1) They further developed the idea that it is the Maillard reaction that results in premature aging and degenerative diseases such as diabetes and heart disease. In this regard, many scientists think the human body may be viewed as a low temperature oven with a relatively long—approximately 75 year —cooking cycle. (2) The Maillard reaction involves a chemical reaction (condensation) between a sugar (usually glucose) with a protein. This complex is known as a Schiff base. In the human body, this is a reversible reaction which reaches equilibrium (i.e., stabilizes) within several hours. With continued exposure to the sugar, the Schiff base undergoes a rearrangement known as non-enzymatic glycosylation that results in a more stable, less reversible substance, known as an Amadori product. Again, in the human body, this process reaches equilibrium over several weeks. (Fig. 1) The Amadori product further degrades irreversibly into a number of highly reactive carbonyl (C=O) compounds. These reactive substances, called Advanced Glycation End products have been designated by the acronym AGE. (3) AGE is a clever pun which reflects the proposed relationship of these reactive substances to aging and age-related diseases. AGEs can further react with other fats, proteins and nucleic acids to form largely indissoluble crosslinks. The age-related accumulation of these AGE products has been demonstrated in many tissues of the body (Fig. 2). (4) Furthermore, during long-term hyperglycemia (elevated blood sugar), as in diabetics, glycation and AGE formation may increase up to four times as much! This explains why diabetics suffer the premature onset of a wide range of age-related complications including cataracts, retinopathy, neuropathy, nephropathy, atherosclerosis and osteoporosis. (5,6)
Crosslinkage Theory Gets New Life
Bjorksten was a talented petroleum chemist. Had he been a food chemist instead, he may have appreciated this link between the Maillard Reaction and crosslinking much earlier, and made even greater progress in developing preventive and therapeutic approaches to crosslinkage-induced aging. Through their insightful work in understanding this process, scientists like Brownlee, Cerami and Monnier provided renewed impetus and a rebirth for the crosslinkage theory. (3) Unfortunately, they did this with little attribution to Bjorksten, who had doggedly pursued this approach to aging for over 50 years.
An important aspect of any comprehensive theory of aging is the inclusion of techniques (dictated by the theory) that can be used to accurately measure the progress of aging. Parameters that can be correlated with age and that can be used to evaluate the rate of aging are known as biomarkers. (7) Scientists have identified a number of unique biomarkers that reflect aging in terms of the Crosslinkage/ Glycosylation theory. (Table 1)
Approaches to Preventing and Removing AGE-Induced Crosslinks
Khalifah and his colleagues proposed a schematic of the formation of AGEs, which illustrates a number of specific therapeutic targets (Fig. 3). ( 8 ) Here are some of the most promising substances to use to inhibit/dissolve AGE-induced crosslinks.
Goat’s rue (Guanidine)
Goat’s rue (Galega officinalis), or French Lilac, has historically been used for the treatment of diabetes since medieval times. The glucose and insulin-lowering effects of Goat’s rue extract are due to the natural substance, guanidine. Guanidine (Fig.4.) is the herbal prototype for the insulin-sensitizing, glucose-lowering anti-diabetic drug, Metformin (Glucophage), and for the related substance, aminoguanidine. The use of raw Goat’s rue is limited in diabetes by its toxicity. The toxicity of Goat’s rue is due to a substance known as galegine (Fig. 5), which can cause reductions in blood pressure and nasal discharge. Vitamin Research’s Goat’s rue extract is a special preparation that is standardized to contain 50% guanidine, with negligible amounts of galegine. CP’s purified, high-guanidine form of Goat’s rue presumably shares most of the beneficial effects of aminoguanidine and Metformin, with none of the adverse effects of raw Goat’s rue herb.
Metformin is an anti-diabetic biguanide that was derived from the herb, Goat’s rue (Galega officinalis). Biguanide drugs were recognized by Prof. Vladimir Dilman as early as the mid-1970s as the most effective anti-aging drugs in existence. Metformin is known as an insulin receptor sensitizer, capable primarily of lowering blood sugar and insulin. Dilman also demonstrated that biguanides restored cortisol receptor sensitivity. Metformin has many other beneficial properties, including optimizing lipid profile, reducing body fat, maintaining levels of growth hormone, stimulating immunity, and extending the maximum lifespan of experimental animals. I reviewed the anti-aging/ life-extending effects of Metformin in the November, 1998 issue of Vitamin Research News. Despite its wide range of reported beneficial effects, Metformin has not, to my knowledge, been tested for its ability to retard AGEs and AGE-induced crosslinks. However, I assume that AGE-inhibiting effects would be found for Metformin, if anyone bothered to look. This situation is analogous to other similar nutrients that have been tested for specific effects, while overlooking effects attributed to their structural cousins. For example, Acetyl-L-Carnitine is used primarily for its cognitive-enhancing, mitochondrial membrane normalizing effects, while L-Carnitine is usually used for its cardiovascular and lipid-normalizing benefits. Dr. Brian Liebovitz, author of L-Carnitine—Vitamin Bt, believes, however, that L-Carnitine is equal to or better as a cognitive enhancer than ALC—its just that no one has ever evaluated the cognitive enhancing effects of L-Carnitine. I think the same could probably be said for Idebenone and its close relative, Coenzyme Q10. They both probably have very similar actions. Likewise, I think Metformin and guanidine (as in CP’s Goat’s rue extract) probably share the AGE and crosslinkage-inhibiting effects of their relative, amino-guanidine. Metformin requires a prescription in the United States.
Aminoguanidine is a substance that has been known for over 100 years. It is structurally very similar to guanidine, the active ingredient in the herb, Goat’s rue. Aminoguanidine has aroused a great deal of interest in the last twenty years, due to its demonstrated ability to block the formation of AGEs and AGE-induced crosslinkages in both animal and human clinical studies. Aminoguanidine inhibits AGE formation, preventing AGE-induced crosslinks in collagen and other tissues. Fortunately, aminoguanidine does not interfere with the formation of normal collagen crosslinks, which are required for structural integrity. Another mechanism by which aminoguanidine is believed to act is by enhancing the action of nitric oxide (the same mechanism by which Viagra functions). (9,10) Aminoguanidine also reduces the formation of lipofuscin (age pigment) and prevents or reduces cataracts, atherosclerosis, diabetic retinopathy, nephropathy and neuropathy (Fig. 6). (2,11-13) In a study with rats, scientists occluded the arteries that supply blood to the brain, inducing an experimental stroke. The scientists administered aminoguanidine in various concentrations and at various time intervals following inducement of the stroke. They found that the size of the brain damage from the loss of blood flow could be greatly reduced with aminoguanidine, even when administered as much as two hours after the onset of the reduction in blood flow. (14) This indicates that aminoguanidine may also be effective in the prevention and treatment of strokes. In one study of diabetic patients, after four weeks of therapy with amino-guanidine, LDL cholesterol decreased almost 30%, and total cholesterol and triglycerides both decreased almost 20%. Hemoglobin-AGE levels, a circulating marker of the degree of glycosylation, also decreased dramatically (13.8 U/mg Hb at the beginning of therapy, to 10.0 U/mg Hb after only four weeks). (15) Although aminoguanidine’s effects on blood sugar and insulin have not been examined, to my knowledge, I believe that if such studies are conducted, the effects will be positive. For example, Metformin and Goat’s rue (guanidine) are best known and best tested for their beneficial effects on blood sugar and insulin, due to their insulin-receptor sensitizing properties. Aminoguanidine, on the other hand, is best known and best tested for its AGE-inhibiting effects. However, I think that if these substances were to be comprehensively evaluated together, we would find that they share most properties, to a greater or lesser degree, due to their closely related structures. Anecdotal reports from patients and physicians appear to confirm this. Aminoguanidine is very safe, as indicated by short-term human studies which used the astronomical dose of 1200 mg daily. (16) (This is in comparison with a usual human dose of 100-300 mg daily). The dose required to cause death in half the animals (mice) to which it was administered (Lethal Dose 50 [LD50]) was 1800 mg/kg.9 That would be equivalent to a human dose of almost 300 gm!
P5P, the active form of vitamin B6, has been found to significantly reduce the nonenzymatic glycosylation (formation of AGEs) of bovine serum albumin (BSA) with radioactive-labeled sugar. Of the substances tested, P5P was exceeded only by aminoguanidine in its ability to inhibit AGE formation (Fig. 7). Combining P5P with guanidine, metformin, or aminoguanidine may enhance their AGE-inhibiting actions even more. (17)
Vitamin B1 (Thiamin)
Alteon is a pharmaceutical company which is focused on developing drugs to prevent the formation of AGE-induced crosslinks, as well as to dissolve crosslinks after they are formed. Several of their products are currently undergoing FDA-sanctioned trials. One of the products, ALT-711, improved arterial elasticity, indicating an ability to undo crosslinkages. (18) This is the first drug that is specifically designed as a crosslinkage breaker. Interestingly, ALT-711 is a derivative of thiamin. In their book, Life Extension, Durk Pearson and Sandy Shaw reported that thiamin was an effective crosslink inhibitor. They were, at that time, consuming two grams of thiamin each day in their personal anti-aging regimens. Thiamin, the parent compound of ALT-711, may ultimately also prove to be an effective crosslinkage breaker as well as inhibitor.
The anti-aging effects of carnosine were detailed in a previous article in the November 2000 issue of Vitamin Research News. Recently, Dr. Alan Hipkiss of the Division of Biomolecular Sciences, King’s College London, reviewed the anti-aging effects of carnosine and aminoguanidine. Dr. Hipkiss believes that one of the major mechanisms of the anti-aging effects of carnosine is its powerful effects as a crosslink inhibitor and breaker, and that the use of these substances might help to control age-related molecular dysfunction.
The venerable crosslinkage theory of aging has clearly gained new respectability in light of the advances in understanding of non-enzymatic glycation and the formation of AGEs and AGE-induced crosslinks. Research in this area is leading to the development of new classes of crosslinkage inhibitors and breakers as anti-aging drugs and nutrients. It is also interesting to note the close relationship between the crosslinkage, neuroendocrine, and free radical theories. Free radicals have been proposed as a cause of crosslinkages, as well as a factor in the loss of sensitivity of receptors of various hormones and neurotransmitters. Also, the loss of insulin receptor sensitivity and impaired glucose metabolism proposed by the neuroendocrine theory, which results in high levels of blood sugar, is clearly a cause/accelerator of crosslinkages. Understanding these processes clearly points at a number of ways to attempt to delay, and in some cases, perhaps even reverse aging. One of the most effective approaches, I believe, is to maintain low levels of glucose and insulin, and minimize the formation of crosslinkage-inducing advanced glycation end products (AGEs). In addition to a low glycemic diet and exercise, I think using either Metformin, aminoguanidine, or Goat’s rue extract, combined with P5P, carnosine, and possibly, additional thiamin, will be found to be a potent anti-aging combination. In the future, crosslinkage breakers like ALT-711 may also become clinically available.