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 cross-links 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 cross-links. 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 imwww.ely solvable approach: using chelating agents to remove toxic heavy metals (especially, aluminum) that were known to be one cause of cross-linking.

He hoped that by eliminating the cross-link-promoting tri-valent (three points of attachment) aluminum atoms (which he believed displaced divalent [two points of attachment]) calcium atoms, he would reduce one of the major sources of cross-linking, and thereby “buy enough time” to solve the rest of the cross-linkage problem.

This has been explained in greater detail in the series of articles on the Cross-linkage Theory of Aging in VRP’s online library at www.vrp.com/art/704.asp, www.vrp.com/art/716.asp, www.vrp.com/art/728.asp, and www.vrp.com/art/765.asp.

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 cross-linkage 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 cross-linking 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 cross-links. These AGE products increase with age in many tissues of the body (Fig. 2).4

Furthermore, if blood sugar remains elevated for prolonged periods (as occurs in poorly controlled diabetics) that may increase glycation and AGE formation up to four times! 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

Cross-linkage 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 cross-linking much earlier, and made even greater progress in developing preventive and therapeutic approaches to cross-linkage-induced aging.

Through their insightful work in understanding this process, scientists like Brownlee, Cerami and Monnier provided renewed impetus and a “rebirth” for the cross-linkage theory.3 Unfortunately, they did this with little attribution to Bjorksten, who had doggedly pursued this approach to aging for more than 50 years.

Cross-linkage Biomarkers: Indicators of Biological Age
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 Cross-linkage/Glycosylation theory (Table 1).

Approaches to Preventing and Removing AGE-Induced Cross-links
Khalifah and his colleagues proposed a schematic of the formation of AGEs, which illustrates a number of specific therapeutic targets (Fig. 3).8 Following are some of the most promising substances to use to inhibit/dissolve AGE-induced cross-links.

Goat’s rue (Guanidine)
Goat’s rue (Galega officinalis), or French Lilac, has 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. We believe Guanidine (Fig. 4) is the herbal prototype for the insulin-sensitizing, glucose-lowering anti-diabetic drug, Metformin (Glucophage), and for the related substance, aminoguanidine.

Metformin (Glucophage)
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.
Dr. Dean reviewed the anti-aging/life-extending effects of Metformin in the November 1998 Vitamin Research News (online at www.vrp.com/library/732342.html). 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 cross-links. However, we 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 (ALC) is used primarily for its cognitive-enhancing, mitochondrial membrane normalizing effects, while L-Carnitine is usually used for its cardiovascular, performance-enhancing, and lipid-normalizing benefits.

Dr. Brian Liebovitz, author of the book, L-Carnitine-Vitamin Bt, believes, however, that L-Carnitine is equal to or better as a cognitive enhancer than ALC—it is just that no one has ever evaluated the cognitive enhancing effects of L-Carnitine. We think the same could probably be said for idebenone and its close relative, coenzyme Q10. They both probably have very similar actions.

Likewise, we think Metformin and guanidine probably share the AGE and cross-linkage-inhibiting effects of their relative, aminoguanidine. Metformin requires a prescription in the United States.

Aminoguanidine (Pimegedine™)
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 cross-linkages in both animal and human clinical studies.

Aminoguanidine inhibits AGE formation, preventing AGE-induced cross-links in collagen and other tissues. Fortunately, aminoguanidine does not interfere with the formation of normal collagen cross-links, 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. 5).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 percent, and total cholesterol and triglycerides both decreased almost 20 percent. 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, we 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, we 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 relatively high doses of 1,200 mg daily.16 (This is in comparison with a usual human dose of 100 to 300 mg daily). The dose required to cause death in half the animals (mice) to which it was administered (Lethal Dose 50 [LD50]) was 1,800 mg/kg.9 That would be equivalent to a human dose of almost 300 gm!

Pyridoxal-5-Phosphate (P5P)
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. 6). Combining P5P with guanidine, Metformin, or aminoguanidine may enhance their AGE-inhibiting actions even more.17

Pyridoxamine
Pyridoxamine (PM) is a third form of B6, and is a well-established inhibitor of AGEs such as CML and CEL (carboxyethyllysine).18 It has recently been discovered that some AGEs, such as CML, can also be derived from lipid peroxidation (“rancid fat”) products.24 These products are called ALEs (advanced lipoxidation end products). PM has been shown in recent studies to block ALE formation, as well.20,21 CML is the predominant AGE in intracellular neurofibrillary deposits in patients with Alzheimer’s disease and in macrophage-derived foam cells in human atherosclerotic plaques. Its concentration in human tissues increases significantly with age.”22

PM has also been shown to block formation of methylglyoxal-AGEs, one of the main AGEs formed inside cells.23 PM reduces methylglyoxal levels in red blood cells and plasma proteins in diabetic rats and prevents formation of the AGE pentosidine in plasma proteins.23

ALT 711, Vitamin B1 (Thiamine) and Benfotiamine
Alteon is a pharmaceutical company that is focused on developing drugs to prevent the formation of AGE-induced cross-links, as well as to dissolve cross-links after they are formed. Several of the company’s products are currently undergoing FDA-sanctioned trials. One of the products, ALT-711, improved arterial elasticity, indicating an ability to “undo” cross-linkages.24 This is the first drug that is specifically designed as a cross-linkage breaker.

Interestingly, ALT-711 is a derivative of thiamine (vitamin B1). In their book Life Extension, Durk Pearson and Sandy Shaw reported that thiamin was an effective cross-linkage inhibitor. Durk and Sandy were, at that time, consuming two grams of thiamine each day in their personal anti-aging regimens. Thiamine, the parent compound of ALT-711, may ultimately also prove to be an effective cross-linkage breaker as well as inhibitor.

Benfotiamine (S-benzoylthiamine-O-monophosphate) is a synthetic, fat-soluble form of thiamine (vitamin B1) that was synthesized in Japan in the 1960s.25,26 BFT has been shown in many human and animal studies to have superior bioavailability to thiamine (B1)—the rate of absorption of therapeutic doses (50 to 100 mg) of B1 is relatively small (usually just four to six percent).27

In the body, both B1 and BFT are transformed into thiamine diphosphate (TDP), the coenzyme form of B1. TDP is essential for the activation of an enzyme called “transketolase.” Transketolase protects cells from AGE formation by diverting the products of glucose metabolism—triose phosphates—into the pentose pathway. If not successfully diverted into this pathway, the triose phosphates become AGEs.28

Comparative studies with B1 and BFT indicate the superiority of BFT absorption. One study found a 120-fold increase in TDP levels in red blood cells from BFT compared to B1,27 and another study with end-stage renal disease patients found that BFT had 430 percent better overall absorption than B1.26 Other scientists reported that “all biokinetic data demonstrated a significantly improved thiamine bioavailability from benfotiamine compared with other preparations.”29

BFT is not only superior to regular B1 in improving TDP-coenzyme B1 status, but it has also been shown to be highly effective in the prevention of AGEs and functional damage in experimental animals30 and humans.31-33

In one study of Type 1 diabetics, doses of BFT of 600 mg per day for 28 days resulted in a 40 percent reduction of red blood cell levels of the AGE, carboxymethyllysine (CML), and 69 percent reduction of intracellular levels of methylglyoxal-derived AGEs.

Significantly, CML levels inversely correlate with diabetic blood vessel damage, and methylglyoxal-AGE is the most important intracellular AGE.31 Other studies of diabetics with peripheral neuropathy showed reduction of pain, increased vibratory sensitivity and normalization of cardiac rhythm.32, 33 A safe and effective human dose of BFT is believed to be approximately 100 mg, taken two or three times daily.

Carnosine
The anti-aging effects of carnosine were detailed in the article in the October 2004 Vitamin Research News (online at www.vrp.com/library/1060676.html). Dr. Alan Hipkiss of the Division of Biomolecular Sciences, King’s College, London, reviewed the anti-aging effects of carnosine and aminoguanidine. He agrees that one of the major mechanisms of carnosine’s anti-aging properties is its powerful effects as a cross-link inhibitor and breaker. He suggested that the combined use of carnosine and aminoguanidine might help to control age-related molecular dysfunction.

Cinnamon Extract (Cinnulin PF®)
Cinnamon has been known to restore insulin sensitivity, normalize blood glucose and insulin levels, and normalize lipid profiles in animals and humans.34-38 Cinnamon Extract (Cinnulin PF®) is a patented cinnamon extract that has even more potent effects than raw cinnamon.39 A 250 mg daily dose of Cinnulin PF divided between two or three meals has been found to help regulate blood insulin, glucose and lipids, and should further help to reduce the formation of AGEs.

Conclusion
The venerable cross-linkage 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 cross-links. Research in this area is leading to the development of new classes of cross-linkage inhibitors and breakers as anti-aging drugs and nutrients. It is also interesting to note the close relationship between the cross-linkage, neuroendocrine and free radical theories. Free radicals have been proposed as a cause of cross-linkages, 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 cross-linkages.

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, we believe, is to maintain low levels of glucose and insulin, and minimize the formation of cross-linkage-inducing advanced glycation end products (AGEs).

In addition to a low-glycemic diet and exercise, we believe a potent anti-aging combination will be found using either Metformin, aminoguanidine, or standardized Goat’s rue extract, along with P5P, pyridoxamine, carnosine, cinnamon extract, plus additional thiamine or benfotiamine. In the future, cross-linkage breakers like ALT-711 may also become clinically available.

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