by Chris Gaugler

I. Description

Lipofuscin (LF) is a conglomerate of lipids, metals, organic molecules, and biomolecules that commonly fluoresces at 360 to 470 nm. The most recent literature concerns a series of syntheses that produce compounds with similar fluorescent properties (345 to 440 nm). Only those that involved the reaction of a lipid (preferentially presenting primary amines), with either an aldehyde or a (transient?) ketone, in the presence of a peroxide yielded stable products fluorescing at the appropriate wavelength. Larger compounds spontaneously formed from initial compounds, and became increasingly stable, while still retaining the same fluorescent element (1).

LF granules have been found in every eukaryote ever examined, and always accumulate within cells as the organism ages, and usually as cellular integrity is challenged. Hence its recognition as "the aging pigment."

II. Research History

Although some researchers believe that LF was identified in the early 1970's (1), the earliest reference to it by the name lipofuscin that I was able to find was in an article on histology of brain cells autopsied in 1937 (2). In 1977 B. L. Strehler described LF-ogenesis as "rare and irreparable metabolic accidents beyond the genetic design result in accumulation of insoluble, non-functional, or noxious by-products of metabolism." (36). In 1981 LF was found to be present in prokaryotes from protozoa to primates (35). As late as 1989 LF was not universally accepted to exist in tissues other than nerve (17). Experiments in 1989 concluded that LF probably was composed of partially degraded mitochondria and ER material rich in metallo-enzymes. These metals, especially iron, were thought to catalyze further oxidative activity including lysosomal degradation (14).

In another 1989 experiment, LF-like substances were found by reaction (in an oxidative environment) of a lipid peroxy free radical with amino acids and proteins and an aldehyde with a primary amine. Attack on tyrosine and subsequent polymerization led to products with the characteristic fluorescence of LF yet of different sizes (9). As late as 1991 LF was thought by some researchers to be generated only in neurons and transported to other cell types (6). A 1993 paper suggested that fluorescence produced by reactions of oxidized lipids with amino acids may be due to polymerization rather than to any structural element (now considered wrong) (10). In 1994 LF was positively identified in cultured muscle and parietal cells, ending the neuron genesis hypothesis (25).

A 1995 paper (Monserrat et al) stated in its introduction that, " Little is known about the saccharide components of situ." In their experiment they tested Vit-E deficient rats for LF content in fat pads and uteri, and also tested human myocytes and neurons. Mannose was the most common sugar residue recovered via delipidation. Also, significant amounts of acetylglucosamine, acetylgalactosamine, sialic acid, galactose, and fucose were recovered. Different lectin binding patterns did occur in different samples, but these reactivity patterns are believed to be transient. They feel that lectin histochemistry may not be an adequate means for ceroid pigment differentiation (29).

III. Characteristics

LF has been positively shown to result from oxidative degradation of mitochondria and/or lysosomes within Purkinje neurons (6). Mercury has been found within LF deposits (3). Aluminum has often been associated with LF fragments (5). Pyrroles are consistently found in LF samples (10). LF always contains iron or copper, and is often associated with zinc or aluminum (19).

A 1992 study concluded that LF-ogenesis is a progressive intracellular process. Characteristic structures, in this case present in macrophages, represented transitional stages between lysosomes and mature pigment granules. It is thought that the residual lipids undergo progressive oxidation secondary to deficient lipolytic activity, overloading the lysosomes (8).

In 1992 Brunk proposed the general mechanism for LF-ogenesis that currently is most accepted. He proposed that LF is formed within secondary lysosomes due to partially reduced oxygen species produced by mitochondria. These may enter the lumen of secondary lysosomes undergoing autophagy and react with iron from degraded cellular structures. This then generates free radicals which induce lipid peroxidation, intermolecular cross-linking, and LF formation (13).

In an vitro experiment, cysteine and iron were needed to initiate mitochondrial peroxidation, "probably through the reduction of ferric to ferrous iron by cysteine with the induction of Fenton chemistry." "Peroxidation could be completely inhibited by desferal, an iron chelator, or butylated hydroxytoluene (BHT), an antioxidant." The typical LF fluorescence of 345-435 nm did not appear for several days, "suggesting that fluorophore is not an immediate product of protein oxidation (22)."

It has also been found that human and synthetic LF have fluorophores in common. This synthesized LF immediately results in formation of singlet oxygen, i.e. LF acts to facilitate "generation of reactive oxygen species that contribute to age-related decline of retinal pigment epithelium (RPE) function and blue light damage (23)."

Paola Timiras determined in 1988 that LF granules consist of lipid (19-51%), protein (30-50%), and acid hydrolysis resistant residue (9-30%) (37). As early as 1967, it was recognized that the protein present in LF must be a structural protein with high lipid-binding tendencies (38).

In an August, 1996 paper, several methods of LF formation and inhibition were examined. Iron-promoted lipid autoxidation, malondialdehyde reactions with amino acids, and the presence of hormones each contributed to LF-ogenesis. The process was inhibited by an increase in pH to 7.4 or by the presence of glutathione or ascorbic acid. Linoleic acid failed to yield a fluorescent product (45).

IV. Interactions and Effects

Age-related macular degeneration (ARMD) is directly correlated (P = .01) with LF pigment accumulation in the basal lamina and Bruch's membrane cells of the retina. Zinc, copper, and iron were found in these deposits (19).

Long-term dialysis treatment can lead to LF-ogenesis in several types of nerve cells. Aluminum is found embedded within the LF granules of dialysis patients (5).

Peroxisomes in epithelial cells of equine efferent sperm ducts appear to proliferate in response to endocytation and oxidation of sperm. Spermatic material cathepsins (natural lysosomal proteases) may break open lysosomes and increase the chances of intracellular oxidation (11). In efferent duct epithelium spermatic material may contribute to LF mass (7).

Iron released by overloaded or dying macrophages may enter other cells and facilitate LDL electrophoretic mobility and LDL oxidation within the cell, starting a LF-ogenic process (12). Modifications of LDL in atherosclerotic lesions, particularly oxidative fusion reactions, contribute to the pathologic process. LF-ogenesis may make these LDL refractory to removal by reverse cholesterol transport mechanisms (15).

Hypoxia damages cellular structures, both physically and chemically (e.g. a reduced ATP concentration). This may make them more susceptible to LF-ogenesis (16).

LF was found in disintegrated lammelar cells of digital pads of aged mice. This result proved that LF accumulation occurs in non-neuronal tissues (18).

Rifabutin treatment for mycobacterial infections increases the rate of LF-ogenesis. Other amphiphilic compounds may induce LF-ogenesis due to their lipid-binding properties (28).

LF was found in the neurons of infants who died without neural pathology and was already apparent at the light microscope level in autopsied neurons of teens. This established that LF-ogenesis was a constant lifespan process (33).

The bizarre findings of the following investigation should be of interest to cell biologists everywhere. This Yugoslavian team found that, "Rat intrascapular brown adipocytes were capable of erythrophagocytosis." Erythrocytes 'squeezed' from the scapular capillary lumen were endocytosed by these brown adipocytes lining the scapula. They could be observed in stages of the process. Some of these erythrocytes 'became' intracellular cells that "...phagocytized adipocyte mitochondria and lipid droplets before their transformation into LF-like bodies or they were degraded into ferritin-like particles observed (on unstained sections) in the mitochondrial matrix, interstitial space, on the periphery of lipid droplets, and in brown adipocyte cytoplasm (49)." These findings certainly raise questions about the accepted competencies of erythrocytes and adipocytes.

Aluminum is consistently present in cells of patients with amyotropic lateral sclerosis (ALS). Samples from nerve cell biopsies of ten ALS patients were examined using particle-induced x-ray emission spectrometry followed by electron energy loss spectrometry. The frequency of characteristic Bunina bodies was tested against nucleolar index, cellular magnesium content, cellular aluminum content, and duration of illness. Aluminum content had the greatest positive correlation. It was determined that aluminum binds strongly to Bunina bodies and to r-ER and also binds to mitochondria and LF, further evidence that aluminum can contribute to intracellular fusion processes (51). Other researchers have found aluminum integrated within LF granules.

In 1995, an experiment in Italy studied the effects of cisplatin treatment on rats. After nine weeks, the numbers of lysosomes and LF granules were 60% higher in the treated group than in the control group (53).

Cultures of retinal pigment epithelial (RPE) cells were infused with either linoleic acid (LA) or linoleic hydroperoxide (LHP). LA from RPE lysosomes was released extracellularly. LHP was not released, was LF-ogenic, and was cytotoxic. RPE cells were incubated with LA or LHP and examined using laser spectroscopy and TEM (55).

Weanling piglets were deprived of selenium and vit-E for two to three months, sacrificed, and their muscle tissue was examined for degenerative alterations. There were extensive LF granules (56).

Buthionine sulfoximine inhibition of glutathione (a naturally occurring antioxidant), in a test on juvenile rats, led to increased LF-ogenesis. Their cellular LF content compared with profiles of aged rats (57).

Inhalation exposure to varying concentrations of dimethylacetamide (DMA), a common by-product of chemical processing, led to marked increases in LF accumulation in rat Kupffer cells. Critical exposure levels were four times the nationally established human safety levels for DMA (59).

Cathepsins are natural lysosomal proteases. The loss of cathepsin activity in aged rats correlates directly with increases in LF (60).

Lead exposure (1% in drinking water) led to accelerated LF-ogenesis in rat pup neuronal and glial cells. Treatment with vimentin to sequester cellular lead resulted in a decline of cellular hypertrophy to near-normal levels (61).

The research cited here indicates that many natural and synthetic compounds can enhance the rate of LF-ogenesis. Chemical exposure effects are so numerous and complex that we may never understand them all, but caution should always be the first response when considering the introduction of any 'new' compound into the environment. On the positive side, there are also means of retarding or preventing LF-osis.

V. Consequences of LF Accumulation

A woman suffering menorrhagia due to endometrial hyperplasia displayed a high rate of LF-ogenesis, causing widespread neuromuscular dysfunction (4). Dementia that is not due to Alzheimer's may be caused by aluminum-associated LF (5). Age-related macular degeneration has a 99% correlation with LF accumulation (19). Creutzfeld-Jacob disease (prions) does not correlate with lipofuscinosis (20).

In both aging and stressed cells, LF correlates with hypoanabolism of RNA and protein, and hypercatabolic accumulation of waste products (21). LF generation of singlet oxygen leading to reactive oxygen species contributes to decline of RPE function (23).

Brain biopsies of a fifteen year old who died of the genetic disease Rett syndrome were examined with light and electron microscopy. LF granules had completely filled cells of the substantia nigra causing increased electron density in the cytoplasm, stacking of ribosomal ER, disorganization of organelles, and evidence of rampant autophagy (26).

The proto-oncogene bcl-2 produces its protein product in direct response to LF accumulation within cells. This protein inhibits generation of reactive oxygen species. This hyper-response could be a contributing factor in the metastasis of some cancers (27). This could occur as bcl-2 activity increases the concentration of bcl-2 protein product in these cells. This raises the probability that carcinogenesis will occur. Rapid division of these 'bcl-2 active' cells is not hindered by the normal concentration of LF that is present in cells where bcl-2 protein levels are lower. This selective advantage helps to speed up cell division and metastasis.

In the genetic condition Batten disease, LF is formed early in life from photoreceptor disc membranes of the RPE. These aggregates cling to the RPE and impede hydraulic efficiency through the retinal basal membrane, reducing blood supply, and leading to early macular degeneration and blindness (30).

Aluminum and silicon found in senile plaques and neurofibrillary tangles of victims of Alzheimer's are actually aluminosilicate components of LF granules coincident in the area and not elements of the lesions themselves. This was determined by fluorescence microscopy, x-ray spectroscopy, and SEM analysis of paraffin sections at autopsy (31).

In autonomic nervous dysfunction concentration of LF correlated well with distension of neural structure. LF accumulation appears to directly affect cell integrity and to disrupt metabolic processes in these cases (34).

In the aging myocardium of rats, LF accumulation has been verified. Oxygen-radical scavenger enzymes are at high levels in this tissue. This lends support to the free radical theory of aging (48).

Cochlear edema causes nerve-pressure deafness. It has been positively correlated with LF accumulation in the subcuticular cytoplasm of hair cells of the cochlea (39).

Imposed epididymal ischemia led to LF accumulation in the epithelial cells and also in macrophages invading the lumina. Epithelial cells in the affected area also displayed increased numbers of lysosomes and engaged in active phagocytosis of spermatozoa (41).

Oxidative stress leads to arrest of the cell cycle in G1 (measured by flow cytometry), an acceleration of telomere degradation from 90 bp per doubling to 500 bp per doubling, and a sharp increase in LF content. These authors propose that LF presence might be contributing to telomere damage and possibly to other failures in accurate genetic replication (42).

A young adult domestic cat, euthanized because of severe neurologic disease, was found to have high levels of LF in neurons of the CNS and PNS. The cat suffered from seizures, blindness, and diminished motor control (43).

LF-osis is associated with some genetic diseases. There are at least seven, and possibly 21 or more such diseases classified as neuronal ceroid lipofuscinoses (52).

VI. Possible Avenues of Intervention

Vitamin E supplements reduced LF-related neuromuscular dysfunction in a woman exhibiting myometrial LF-osis. Her speech and mobility both improved significantly (4).

The Romanian team of Riga and Riga, from 1972 to 1992, worked on creating an orally administered antagonist to LF. They were able to develop "...a neurometabolic antioxidative therapy, finally represented by a specific antistress and antiaging synergistic formula...Its homeostatic actions have been demonstrated in the stressed aging brains by reestablishing the anabolism/catabolism balance: anabolic regeneration by increasing total RNA, total proteins and water-soluble proteins, coupled with catabolic regulation by accelerated LF-olysis and age pigment elimination (neuronal-->glial-->capillary route) and decreasing of water-insoluble proteins." Successful human testing has been done on this formula (21).

In a test on Sprague-Dawley rats varying amounts of pyruvate, vit-E, and clofibrate (a peroxisome proliferator and free radical enhancer) were administered to eleven test groups. Pyruvate prevented liver swelling. Vit-E or pyruvate prevented abnormal LF-ogenesis. Pyruvate increased plasma concentrations of the HNO metabolites NO and NO , leading to a reduction of per oxidative free radical activity (32).

Increased concentrations of the antioxidant indenoindole, (+20 uM) and (+40 uM), decreased LF-ogenesis in cultured rat myocardial cells by 18% and 24% respectively (40). This was one of a continuing series of experiments that are examining oxygen radical-related diseases.

"Physiological levels of spermine reduce LF accumulation by 20%, and that antioxidative effect compares with that of vit-E ...spermine prevents accumulation of free iron in myocytes, probably acting as a chelating agent." The effect compares to that of deferoxamine (a prescription iron chelator) (46).

Dietary restriction to near-minimal survival levels virtually prevents LF-ogenesis. Age reduces both aldehyde dehydrogenase and glutathione-S-transferase activities, but restricted animals retained these activities, and LF accumulation was much reduced (50).

Methods of increasing glutathione (a natural antioxidant) production (creating a hostile environment to LF) could retard neuronal accumulation of LF (57). This might be accomplished through supplements or even using gene therapy.

Rainbow trout given vit-E in dosages of 200 mgE/kg food were examined at death and after eight months of cold storage at -18 C. No peroxides were detectable at any time (too low to measure?), malondialdehyde was significantly reduced, LF concentrations were lower, and fatty acid concentrations were lower in the crude fat (58).

VII. Conclusions

Lipofuscin is a name assigned to a class of intracellular compounds that fluoresce at approximately 400 nm. The common elements responsible for this compound apparently must include 1) a metal or metalloid, 2) a lipid or protein fragment with an oxidizable site (e.g. a hydroxyl, sulfhydryl or amide), 3) a peroxide (or perhaps another strongly oxidative molecule), intracellularly H O is most likely to be involved, 4) an aldehyde or transient ketone body, and 5) reduction in integrity of cellular elements and/or a low level of antioxidants. LF seemingly coalesces about a structure of consistent character that always contains iron or aluminosilicate or another metal or metalloid. The presence of this organometallic compound accelerates further oxidative events within the cell until granules accumulate that interfere with the proper structures and activities of the cell. Non-dividing cells are at greatest risk as dividing cells have a chance of leaving LF granules in one daughter cell (becoming non-viable) while the other daughter cell is left relatively 'clean.'

Standard symptoms of LF-osis relate to the tissues that are affected. Neurologic and neuromuscular disorders are most common, with muscular dysfunctions a distant second. Palsy, dementia, weakness, malabsorption and organ failure may occur.

A lowered metabolism, restricted diet, avoidance of oxidants, infusion of antioxidants or other metabolites, and a clean environment (free of toxins and stress) all contribute to amelioration of LF-osis. It is likely that prevention of LF-osis is possible now or in the very near future, even though there is not yet a universal consensus as to what LF is.

VII. References

Copyright 1997 Chris Gaugler and Koni Stone

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