Eduardo A. Porta

Pigments in Aging: An Overview

Although during the normal aging process there are numerous pigmentary changes, the best recognized are those of melanin and lipofuscin. Melanin may increase (e.g., age spots, senile lentigo, or melanosis coli) or decrease (e.g., graying of hair or ocular melanin) with age, while lipofuscin (also called age pigment) always increases with age. In fact, the time‐dependent accumulation of lipofuscin in lysosomes of postmitotic cells and some stable cells is the most consistent and phylogenetically constant morphologic change of aging. This pigment displays a typical autofluorescence (Ex: ∼440; Em: ∼600 nm), sudanophilia, argyrophilia, PAS positiveness, and acid fastness. Advances on its biogenesis, composition, evolution, and lysosomal degradation have been hampered by the persistent confusion between lipofuscin and the large family of ceroid pigments found in a variety of pathological conditions, as evidenced by the frequent use of the hybrid term lipofuscin/ceroid by investigators mainly working with in vitro systems of disputable relevance to in vivo lipofuscinogenesis. While lipofuscin and ceroid pigments may share some of their physicochemical properties at one moment or another in their evolutions, these pigments have different tissue distribution, rates of accumulation, origin of their precursors, and lectin binding affinities. Although it is widely believed that lipofuscin is a marker of oxidative stress, and that it can be, therefore, modified by antioxidants and prooxidants, these assumptions are mainly based on in vitro experiments and are not generally supported by in vivo studies. Another common misconception is the belief that lipofuscin can be extracted from tissues by lipid solvents and measured spectrofluorometrically. These and other disturbing problems are reviewed and discussed in this presentation.

Sequential histochemical studies of neuronal lipofuscin in human cerebral cortex from the first to the ninth decade of life

The typical and most consistent physico-histochemical properties of lipofuscin granules, such as autofluorescence, sudanophilia, acid-fastness, PAS-reactivity, and lectin reactivities for diverse saccharide moieties have been generally detected in tissue specimens of old humans and animals. The purpose of this study was, therefore, to explore possible sequential variations of each of these properties in cortical neurons of the left cerebral temporo-parietal areas from individuals dying from the first to the ninth decade. Autofluorescence was studied with an ad hoc equipped microscope, sudanophilia was evaluated by Oil-red-O (ORO) staining, acid-fastness by long Ziehl-Nielsen reagent, PAS reactivity by the periodic-acid-Schiff reagent before and after diastase treatment, and the saccharide moieties by the use of a commercial kit of seven different biotinylated lectins. In the specimen from a 5-year-old child, lipofuscin granules were detected in less than 5% of the cortical neurons, but these granules already showed golden-yellow autofluorescence, sudanophilia, acid-fastness and PAS-reactivity. From the second to the ninth decade of life, perikaryal lipofuscin granules were found in practically all cortical neurons with apparent agewise increases in the intensity of sudanophilia and PAS-reactivity, but with variable acid-fastness expression. Surprisingly, however, no saccharide residues were detected by lectin histochemistry before the fifth decade of life. First detected saccharide was mannose in specimens from the fifth decade of life, and at later decades acetyl galactosamine, sialic acid and lactose were also found. Although, the reasons for the absence of lipofuscin affinity for the seven lectins used in this study in the cortical neurons of young and middle-aged individuals are presently unknown, these unexpected findings suggested important evolutionary changes of biogenesis and composition of the age-pigment.

Recent advances in molecular pathology: a review of the effects of alcohol on the liver.

Since the recognition of the frequent association of human chronic alcoholism with fatty liver and cirrhosis in the middle of the nineteenth century (Addison, 1836; HUSS, 1849; Rokitansky, 1849), there has been a constant effort by many investigators to clarify the pathogenesis of these hepatic lesions. It was only natural that researchers in this area had tried to reproduce in animals the spectrum of lesions usually encountered in man. A great deal of experimental work has concentrated in the study of the acute effects of a single dose of ethanol on the liver. By this approach, transient fatty livers and certain ultrastructural alterations were readily induced in rats. From these investigations, a fairly complete picture of the metabolic changes occurring during ethanol oxidation has emerged. Despite these advances, the pathogenesis of the acute morphologic hepatic changes is still unresolved. For those investigators more interested in the multifaceted type of lesion associated with the chronic consumption of large amounts of alcohol, the experimental work was plagued with difficulties and failures. When rats were offered alcohol for prolonged periods in the drinking water, separately from the solid food, they consumed relatively low amounts of alcohol and developed only fatty livers and some hepatofibrosis if the final diets (alcohol + food) were grossly inadequate (Best et al., 1949). This situation generated confusion and controversy, particularly in regard as to whether or not alcohol acted as a hepatotoxic independently of dietary factors. Fortunately, in the last decade most of the difficulties in the chronic models of alcoholism in rats have been successfully solved by two newly devised methods of administering the alcohol and by more carefully controlled nutritional approaches (Lieber et al., 1963; Ports and Gomez-Dumm, 1966, 1968). The reproduction of all the hepatic lesions of the human alcoholic, as well as their prevention and regression by simple dietary means, have been recently achieved in rats. As a result the interrelations of alcohol and food have been greatly clarified (Gomez-Dumm and Porta, 1966; Gomez-Dumm et al., 1968; Hartroft and Porta, 1966, 1967, 1968; Hartroft et al., 1966, 1969; Jabbari and Leevy, 1967; Jones and Greene, 1966; Koch et al., 1968; Lieber and De Carli, 1966; Porta, 1969; Porta and Gomez-Dumm, 1966, 1968; Porta and Hartroft, 1965a; Porta et al., 1965a, 1966, 1967, 1968, 1969a, b, c). While controversial views are likely to persist for awhile yet in the field of acute and chronic alcoholism, it is our intention to review here experimental

Prooxidant and antioxidant hepatic factors in rats chronically fed an ethanol regimen and treated with an acute dose of lindane

While acute lindane treatment and chronic ethanol feeding to rats have been associated with hepatic oxidative stress, the possible roles of these stresses in the pathogenesis of hepatic lesions reported in acute lindane intoxication and in those observed in some models of chronic alcoholism have not been established. Our previous studies in rats chronically fed ethanol regimens and then treated with a single intraperitoneal (i.p.) dose of lindane (20 mg/kg) showed that while lindane per se was invariably associated with hepatic oxidative stress, chronic ethanol feeding only produced this stress when the dietary level of vitamin E was relatively low. Chronic ethanol pretreatment did not significantly affect the lindane-associated oxidative stress, and neither chronic ethanol feeding nor acute lindane, single or in combination, produced any histologic and biochemical evidence of liver damage. In the present experiment, the acute dose of lindane was increased to 40 mg/kg, and we have studied a larger number of prooxidant and antioxidant hepatic factors. Male Wistar rats (115.5 +/- 5.4 g) were fed ad lib for 11 weeks a calorically well-balanced and nutritionally adequate basal diet, or the same basal diet plus a 32% ethanol/25% sucrose solution, also ad lib, and were then injected i.p. with a single dose of lindane or with equivalent amounts of corn oil. The results indicated that acute lindane treatment to naive rats increased practically all the prooxidant hepatic factors examined (cytochromes P450 and b5, NADPH cytochrome c reductase, NADPH oxidase), as well as the generation of microsomal superoxide radical and thiobarbituric acid reactive substances of liver homogenates, but did not modify any of the antioxidant hepatic factors studied. Conversely, the chronic administration of ethanol alone did not significantly affect the prooxidant hepatic factors but reduced some of the antioxidants (i.e., the activities of GSH-Px and the contents of alpha-tocopherol and ubiquinols 9 and 10). Although chronic ethanol pretreatment further increased the superoxide generation induced by lindane per se, it did not increase but generally reduced the effects of lindane per se on the other prooxidant factors studied. Furthermore, although acute lindane administration to ethanol-pretreated rats was associated with decreases in GSH and catalase (not affected by ethanol or lindane treatment alone), it did not substantially modify the reducing effects of ethanol feeding per se on GSH-Px, alpha-tocopherol, and ubiquinols. Once again, neither chronic ethanol feeding nor lindane treatment, single or in combination, was associated with any evidence of liver damage.

CHANGES IN CATHEPSIN B AND LIPOFUSCIN DURING DEVELOPMENT AND AGING IN RAT BRAIN AND HEART

While results with inhibitors of thiol proteases have led to the suggestion that the progressive increase with age of lipofuscin in post-mitotic and some stable cells may be due to an age-related decline in the activity of these enzymes (Ivy et al., 1989), no direct evidence has been yet presented to support this hypothesis. In this study Wistar female rats were killed at age of 5, 14, and 24 months and the amounts of lipofuscin were histologically quantitated in neurons of the left cerebral parietal cortex and in cardiac myocytes of left ventricle. The sites of cathepsin B activity histochemically detected were quantitated in sections from left cerebral parietal cortex and left ventricle, and the activity of this enzyme was also measured biochemically in brain and heart homogenates. In line with previous findings, the amounts of lipofuscin in neurons and cardiac myocytes increased linearly during development and aging (from 5 to 14 and from 14 to 24 mo.). The sites of cathepsin B activity histochemically detected in sections from cerebral cortex significantly increased from 5 to 14 mo., but remained unchanged from 14 to 24 mo, while in sections from the left cardiac ventricle these sites of activity remained unchanged during development, and significantly increased during aging. On the other hand the biochemically determined activities of cathepsin B in brain and heart homogenates remained unchanged from 5 to 14 mo., but significantly decreased from 14 to 24 mo. These results suggest that the increase in lipofuscin with age may not be due to an age-wise decline in cathepsin B activity.

Lectin histochemistry of lipofuscin and certain ceroid pigments

Little is known at present about the saccharide components of lipofuscin (age pigment) and ceroid pigments in situ. The purpose of this study was, therefore, to study in detail the lectin reactivities of lipofuscin in neurons and cardiac myocytes of old humans and rats. In addition, those of diverse ceroid pigments found in human aortic atheromas, in the livers of choline-deficient rats, in the uteri of vitamin E-deficient rats and in the crushed epididymal fat pad of rats, are included. Cryostat and deparaffinized sections from all these tissues were either extracted with a solvent mixture of chloroformmethanol-water (10∶10∶3, v/v) and incubated with 7 different biotinylated lectins or left untreated. Delipidation was done in order to study whether it was possible to discriminate between the saccharide moieties of glycolipids and glycoproteins of lipofuscin and ceroid pigments in situ. Other similarly treated sections were used to study the autofluorescence, sudanophilia, acid-fastness and reactivity to PAS. The frequency and intensity of lectin binding and standard histochemical properties of all the pigments were evaluated semi-quantitatively and blind. The results indicated that mannose was in general the most consistently detected sugar residue in lipofuscin granules of humans and rats, and that this pigment may also contain acetylglucosamine, acetylgalactosamine, sialic acid, galactose and fucose. However, notable differences were found not only in the lipofuscin saccharide components of different cell types of humans and rats, but also in those in the same type of cells in both species. Although mannose was not detected in the hepatic ceroid of choline-deficient rats, this saccharide moiety was almost always present in the other ceroid pigments. Each of the ceroids also contained other types of saccharides although the frequency of the latter varied between different ceroid pigments. While lipofuscin and each of the ceroid pigments showed somewhat different lectin binding patterns, the variability in the frequency of reactivity to lectins suggests that these patterns may not be permanent but transient. In this sense, it appears that lectin histochemistry may not allow these pigments to be differentiated. Furthermore, the extractive procedures used in this study did not enable us to determine whether the saccharides detected in the pigments in situ corresponded to glycolipids or glycoproteins.

Effects of dietary protein levels on the Saimiri sciureus.

Abstract Despite the increasing use of the squirrel monkey in nutritional studies, little is known of the dietary requirements of this species. Twelve young (700 gm initial wt) male squirrel monkeys were allotted to four groups of three animals each, and were offered ad libitum, during 24 weeks, isocaloric diets in which the amounts of protein were 25%, 12.5%, 9%, or 6%. The dietary protein employed was a purified soya protein and was supplemented with 2.1 gm% of methionine to obtain the same content of this amino acid as present in casein. The diets contained amounts of vitamins and essential food factors in excess of the estimated normal requirements. The results indicate that the diets with 25% protein permit the growth and the normal maintenance of several functional and morphologic parameters studied. An almost similar effect was observed with 12.5% protein diets. The protein intake in monkeys fed this diet was 8 gm/day/kg of body weight, and it appears to be the minimal normal protein requirement for this species in captivity under the experimental conditions employed in our work. The effect of 9% and 6% protein diets was manifested by poor ponderal growth, decrease in albumin/globulin ratio, decrease in hemoglobin concentration, decrease in BUN, and increase in serum alkaline-phosphatase activity. In addition, the livers of animals on the low-protein diets had moderate to severe ultrastructural alterations.

Role of lipoperoxidation in early choline deficiency

Abstract Although a significant increase in hepatic triglycerides and an alteration in the relative lecithin contents of hepatic mitochondria were found in rats 12 hours after the institution of a cholinedeficient regimen, neither in their mitochondria nor in their microsomal fractions could diene conjugation be detected at 3, 6, 12, or 120 hours. It appears, therefore, that lipoperoxidation is not a factor in the development of hepatic changes associated with early choline deficiency.

Differential lectin histochemical studies on lipofuscin (age-pigment) and on selected ceroid pigments

The persistent indiscriminate use of the term lipofuscin for the pigments encountered in pathological conditions, and which should be most properly termed ceroid pigments, is still creating unnecessary conceptual and nomenclature problems, and a great deal of confusion. While both the age-dependent lipofuscin and the pathologically formed ceroid pigments have somewhat similar physical and histochemical properties, sufficient differences to properly identify these two types of pigments are presented in this communication. In addition, because little is known on the saccharide components of lipofuscin and ceroid pigments in situ, we have in recent years explored the lectin binding characteristics of lipofuscin in human and rats, as well as in diverse ceroid pigments experimentally induced in rats. Our lectin histochemical results showed qualitative and quantitative differences in the saccharide composition between human cerebral neurolipofuscin and the intra and extracellular ceroid pigment of human atheromas, as well as, between rat lipofuscin and the ceroid pigments induced in these animals.

Dietary factors in lipofuscinogenesis and ceroidogenesis

The presence of ceroid pigments in human and animal tissues is associated with numerous pathological conditions in which the main pathogenic factor is the primary or secondary deficiency of vitamin E or imbalances between anti- and pro-oxidants. That oxidative stress, particularly through its consequent lipid peroxidation, plays a capital role in the genesis of ceroid pigments, is supported by numerous in vitro and in vivo studies. Discussed in this presentation are two examples of oxidative stress on ceroidogenesis, namely the in vivo rat model of dietary hepatic necrosis, and the in vitro formation of ceroid pigments by the aerobic incubation of unsaturated fat and blood cells. Although it is widely believed that the progressive accumulation of lipofuscin is also a marker of oxidative stress, and that this pigment can be modulated by the dietary anti- and pro-oxidant factors, the evidence for these related notions is highly questionable. Some years ago, this controversial problem was reexplored in our laboratories by a series of studies in Wistar male rats, and the results indicated that neither the type of dietary fat, nor the pharmacological amounts of vitamin E significantly influenced the amounts of lipofuscin in cerebral neurons, cerebellar Purkinje cells, hepatocytes or cardiac myocytes. It was also found that the indices of lipid peroxidation determined in this study (production of malonaldehyde, and detection of conjugated dienes) did not correlate with the progressive accumulation of lipofuscin with age. All these results strongly suggest that the presence and cellular accumulation of lipofuscin can hardly be considered a marker of oxidative stress.