Lipofuscin (aging) pigment granules of the newborn human liver.

We have observed pigmented cytoplasmic granules, with the characteristic staining properties of lipofuscin (ceroid, "wear-and-tear") pigment, in newborn human liver. The pigment is found at the periphery of the lobule in hepatocytes and some bile ductular cells. It is acid-fast, PAS-positive after diastase digestion, slightly argyophilic and sudanophilic, and markedly Schmorl's- and peroxidase positive in paraffin sections. Difficult to see in sections stained with hematoxylin and eosin, the pigment can be detected in unstained sections. The granules also resemble lipofuscin found in adult tissues, in their ultra-structural and enzymatic properties. They are polymorphic, contain granular material of moderate and high electron opacity, and are delimited by a single membrane. Acid phosphatase and beta-glucuronidase activities are visualized in the newborn granules, identifying them as lysosomes. The granules also contain copper and, to a much lesser extent, iron. The accumulation of lipofuscin pigment in lysosomes in many tissues correlates well with aging, and this process has been interpreted as a reflection of cellular degeneration or wear-and-tear. However, the presence of lipofuscin granules as a constant component of neonatal liver suggests that they are not a measure of cellular senescence.

Ultrastructural and cytochemical demonstration of peroxisomes in cultured fibroblasts from patients with peroxisomal deficiency disorders.

The oxidation of very long chain fatty acids and synthesis of ether glycerolipids (plasmalogens) occurs mainly in peroxisomes. Zellweger's cerebrohepatorenal syndrome (CHRS) is a rare, inherited metabolic disease characterized by an apparent absence of peroxisomes, an accumulation of very long chain fatty acids, and a decrease of plasmalogens in tissues and cultured fibroblasts from these patients. As peroxisomes are ubiquitous in mammalian cells, we examined normal and CHRS-cultured fibroblasts for their presence, using an electron microscopic histochemical procedure for the subcellular localization of catalase, a peroxisomal marker enzyme. Small (0.08-0.20 micron) round or slightly oval peroxisomes were seen in both normal and CHRS fibroblasts. The number of peroxisomes was analyzed morphometrically and found to be significantly reduced in all CHRS cell lines. These results are discussed in relation to the underlying defect in peroxisomal function and biogenesis in this disease.

Hemoglobin uptake by rat hepatocytes and its breakdown within lysosomes.

The peroxidatic activity of hemoglobin permitted visualization of its uptake by rat hepatocytes by means of the Graham-Karnovsky 3,3'-diaminobenzidine (DAB) procedure. Lysosomes were visualized by their acid phosphatase, beta-glucuronidase, and glucosaminidase activities. When large doses of rat, cow, or human hemoglobin are intravenously injected, or when hemoglobinemia is induced by injection of distilled water, DAB-positive hemoglobin is engulfed by pinocytosis. Pinocytotic vacuoles become digestive vacuoles ("phagolysosomes") by fusion with lysosomes of the dense body type that have moved from their pericanalicular position. By 16-24 hr after even massive amounts of hemoglobin (400 mg/100 g), the protein is barely demonstrable in hepatocytes. At the lowest doses of injected hemoglobin (15 mg/100 g body weight), DAB-positive vacuoles are demonstrable only in the Kupffer cells.

Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.

Hypolipidemia in a mutant strain of "acatalasemic" mice.

Mutant "acatalasemicm" mice have an unstable catalase in hepatic and renal peroxisomnes that is readily degraded, apparently into peroxidase subunits. The hypothesis that an in situ cataloperoxidase would affect serum lipids was tested in these mutants and serum triglycerides and cholesterol were found to be significantly lower than in the wild strain. This finding is in accordance with reports that a hypolipidemic response to the injection of peroxidase subunits of hepatic catalase occurs in humans and rabbits.

Peroxisomal defects in neonatal-onset and X-linked adrenoleukodystrophies.

Accumulation of very long chain fatty acids in X-linked and neonatal forms of adrenoleukodystrophy (ALD) appears to be a consequence of deficient peroxisomal oxidation of very long chain fatty acids. Peroxisomes were readily identified in liver biopsies taken from a patient having the X-linked disorder. However, in liver biopsies from a patient having neonatal-onset ALD, hepatocellular peroxisomes were greatly reduced in size and number, and sedimentable catalase was markedly diminished. The presence of increased concentrations of serum pipecolic acid and the bile acid intermediate, trihydroxycoprostanic acid, in the neonatal ALD patient are associated with a generalized diminution of peroxisomal activities that was not observed in the patient with X-linked ALD.

Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome.

The cerebro-hepato-renal syndrome is a rare familial malady with cerebral, renal, and skeletal abnormalities, severe hypotonia, cirrhosis, iron and lipid storage, and death within 6 months. Correlated electron microscopic, histochemical, and biochemical studies demonstrate defects in two oxidative organelles. Peroxisomes cannot be found in hepatocytes and renal proximal tubules. In hepatocytes and cortical astrocytes, mitochondria are distorted in their appearance and glycogen stores are increased. Oxygen consumnption of brain and liver mitochondrial preparations with succinate and with substrates reducing nicotinamide adenine dinucleotide is markedly diminished, but the consumption is normal with ascorbate and tetramethylphenylenediamine, which suggests a defect in electron transport prior to the cytochromes. Histochemical studies of mitochondrial oxidation point to a defect between the succinate dehydrogenase flavoprotein and coenzyme Q, possibly in the region of nonheme iron protein.

Hypophosphatasia: clinicopathologic comparison of the infantile, childhood, and adult forms.

Histochemical and direct enzyme analysis of osseous tissue from 23 patients with hypophosphatasia revealed that all clinical forms of this inherited metabolic bone disease are characterized by deficiency of alkaline phosphatase (ALP) activity in bone. The severe infantile form has the most profound deficiency, infantile form has the most profound deficiency, yet the cellular source of this enzyme--osteoblasts and their matrix vesicles--are normal by routine light and electron microscopy. Despite radiographic changes in bone metaphyses consistent with rickets, iliac crest biopsy of one affected child revealed no abnormalities; the other had evidence of a mineralization defect, but not as severe as that in affected infants. In this child and several affected adults with osteomalacia, osteoblasts appeared flat and metabolically inactive. Although these histological changes suggested a different pathogenetic mechanism for adult and childhood hypophosphatasia, these changes are most likely secondary to the underlying osteomalacia. Our findings are most consistent with evidence that childhood and adult hypophosphatasia often represent clinical expression of the heterozygous state for ALP deficiency which, when homozygous, results in the clinically severe, recessive, infantile form. Histochemical and direct analysis of bone tissue from controls and patients with hypophosphatasia demonstrated that the severe infantile form is associated with the most severe ALP deficiency. In the milder clinical forms, ALP deficiency in bone is not as profound. In general, the severity of the clinical expression of hypophosphatasia reflects the magnitude of the deficiency of ALP in bone. This is the expected finding for this inborn error of metabolism, which illustrates the major role bone ALP activity has in the process of normal skeletal mineralization.

Peroxisomes in disease.

Cytochemical, biochemical and morphological changes in peroxisomes have been described in human metabolic disorders, in experimental models of disease and in response to drugs and toxins. These include the cerebrohepatorenal syndromes, in which peroxisomes can not be detected and mitochondrial respiration is inhibited, atherosclerosis, alcoholic cardiomyopathy, and tolerance to oxygen toxicity. Although information on the role of peroxisomes in disease is limited, increased awareness of their widespread distribution and the availability of an improved cytochemical procedure for staining peroxisomes in human specimens should provide new insights into their function.

The internal reticular apparatus of Camillo Golgi: a complex, heterogeneous organelle, enriched in acid, neutral, and alkaline phosphatases, and involved in glycosylation, secretion, membrane flow, lysosome formation, and intracellular digestion.

Its topography is one of the most characteristic features of the Golgi apparatus and the reticular nature of this organelle is evident in Golgi's first drawings, in light microscopic enzyme cytochemical preparations, and in high voltage electron micrographs of thick sections. Although individual components of the Golgi apparatus may differ in staining characteristics, morphology, contents, and enzymatic activities, they are integrated into a dynamic topographical and functional unit that is closely associated with the endoplasmic reticulum. Modulation of enzymatic activities and morphological and enzymatic heterogeneity are not surprising in an organelle that is the site of both synthetic and digestive events, including glycosylation, sulfation, formation of secretory granules and lysosomes, and the degradation of endocytized material.

Cytochemistry of human catalase. The demonstration of hepatic and renal peroxisomes by a high temperature procedure.

The cytochemical demonstration of marker enzymes for subcellular organelles permits light microscopic analysis of their structure and function in normal and diseased tissues. Currently available staining procedures for the peroxidatic activity of catalase in peroxisomes of plant and animal cells yield weak and inconsistent light microscopic staining when applied to human tissues. We have developed a simple and sensitive high temperature procedure that clearly and reproducibly stains these abundant, but poorly understood, organelles in biopsy specimens of human liver and kidney. This method utilizes formaldehyde fixation, a modified diaminobenzidine (DAB) medium, incubation at 45 degrees C and postosmication for both light and electron microscopy.

Primary fixation in osmium-potassium ferrocyanide: the staining of glycogen, glycoproteins, elastin, an intranuclear reticular structure, and intercisternal trabeculae.

Primary fixation in an osmium-potassium ferrocyanide (K4Fe(CN)6) mixture combines selective fixation, staining, and extraction of various cellular components; membranes, glycogen, glycoproteins, and elastin are preserved and stained. An intranuclear reticular structure that is composed of 3-6 nm fibers and permeates the entire nucleus, except for the nuclear pores, is demonstrated by electron microscopic examination of tissues prepared in an osmium-potassium ferrocyanide fixative. Condensations of the reticulum parallel the distribution of heterochromatin in interphase nuclei. This preparative procedure also reveals a network of trabeculae that are associated with the cisternae of rough endoplasmic reticulum and connect the parallel cisternae in hepatocytes, plasmacytes, neurons, and pancreatic ancinar cells. The intercisternal trabeculae are associated with both free and bound ribosomes.

Ultrastructure and staining properties of aortic microfibrils (oxytalan).

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.

Extracellular matrix microfibrils are composed of core proteins coated with fibronectin.

Extracellular proteins of cultured calf aortic smooth muscle cells consist predominantly of microfibrils 10-20 nm in diameter typical of "elastin-associated" microfibrils described in many tissues. Chemical and immunochemical evidence is presented that microfibrils consist of at least two proteins: core protein and fibronectin. Insoluble proteins of the microfibrils were obtained in the form of a pellet and antibodies raised in rabbits against these components. The antisera reacted with the insoluble microfibrillar proteins and with soluble fibronectin in enzyme-linked immunosorbent assay, and immunostained the extracellular microfibrils in cultured cells. An immunoglobulin (Ig) fraction was prepared and absorbed with fibronectin. The absorbed IgG retained its reactivity with the microfibrillar proteins but was no longer reactive with soluble fibronectin. Immunofluorescence studies were carried out using the absorbed IgG and IgG to soluble fibronectin. Both antibodies showed immunoreactive microfibrils in the extracellular matrix of cells in log phase. However, with increasing time in culture, as the cells reached confluence, the immunofluorescence of microfibrils reacting with the absorbed IgG became less intense, whereas that of microfibrils reacting with IgG to fibronectin increased; in confluent cells, essentially no staining was detected with the absorbed IgG, and a dense network of intensely stained microfibrils was seen with IgG to fibronectin. Treatment of these cultures with urea led to partial dissociation of the fibronectin and increased visualization of the microfibrils with the absorbed IgG; double-label immunofluorescence showed that both proteins occurred on the same microfibrils. The localization of immunoreactive sites to the extracellular microfibrils was confirmed by immunoelectron microscopy. Nearly quantitative cleavage with CNBr failed to dissociate the antigenically active fragments of fibronectin from the CNBr fragments of the core proteins of the microfibrils. It was concluded that microfibrils contain core proteins and fibronectin that are codistributed in insoluble, possibly covalently cross-linked, aggregates. The core proteins are first deposited by the cell and, as a function of time in culture, fibronectin gradually coats their surface.

Immunocytochemical localization of hepatic legandin and Z protein utilizing frozen sections for light and electron microscopy.

Ligandin (glutathione-s-transferase) and Z protein are soluble hepatocellular proteins that are involved in the transfer of organic ions, including bilirubin and some hormones and carcinogens from the plasma to the liver. The intracellular distribution of ligandin and Z protein was studied by applying the peroxidase-antiperoxidase procedure of L. A. Sternberger (Immunocytochemistry, Prentice Hall Inc., 1974) to paraffin sections and free-floating 10-micrometers frozen sections that were processed for both light and electron microscopy. Ligandin and Z protein were localized to the cytosol of hepatocytes in association with smooth endoplasmic reticulum (SER), but no reaction product was present between cisternae of rough endoplasmic reticulum. Penetration of reagents was enhanced in 10-micrometers frozen sections and the preservation of subcellular structures was equivalent to thicker, unfrozen sections.

Peroxisomes (microbodies) in human liver: cytochemical and quantitative studies of 85 biopsies.

The number, intracellular distribution, and staining characteristics of human hepatocellular peroxisomes that had been made visible by cytochemical staining for catalase were evaluated in biopsies from 75 patients with hepatic, inflammatory, or malignant disease and ten normal individuals. Intensity of staining was found to be proportional to enzymatic activity by microspectrophotometry. Scanning transmission electron microscopy (STEM) image analysis demonstrated an inverse relationship between peroxisomal size and contrast. Peroxisomes were more abundant, and often concentrated in a perinuclear configuration in cholestatic and cirrhotic livers. Decreased peroxisomal staining was common in cholestasis, cirrhosis, hepatitis, and in almost all patients with malignancies, both with and without hepatic metastases.

Lysosomes and the sclerotic arterial lesion in Hurler's disease.

A case of Hurler's disease in a mentally retarded, six year old boy is reported. In Hurler's disease a lysosomal hydrolase, l-iduronidase, is deficient, and consequently undegradable mucopolysaccharide accumulates within lysosomes in many tissues. Severe occlusive coronary artery disease and sclerotic aortic lesions are common in very young patients, although their serum lipid and blood pressure levels are normal. Vascular collagen and elastin is increased, but little or no stainable lipid is present. Electron microscopy shows that aortic smooth muscle cells are distended by vacuoles, appearing empty in formalin fixed tissues, that identify them as the "gargoyle" cells in the proliferative lesion. The presence of a basic lysosomal defect and the absence of other contributing metabolic factors suggest that accumulation of an excess of undegradable substrate within smooth muscle lysosomes may be an initiating event in the development of proliferative sclerotic vascular lesions.

Microfibrillar cardiomyopathy: An infiltrative heart disease resembling but distinct from cardiac amyloidosis.

Microfibrils-small, ubiquitous components of the extracellular matrix in many tissues-generally have not been recognized as causing infiltrative heart disease, except in a group of cardiac transplant patients treated with cyclosporin. Microfibrils are often associated with elastic tissue and contain the glycoprotein fibrillin, the P component of amyloid, and bound fibronectin. A genetically determined abnormality of fibrillin caused by point mutations of fibrillin genes recently was reported as the cause of Marfan's syndrome. However, to date, no abnormalities of increased fibrillin tissue deposition have been observed. In the last two years, while examining right ventricular endomyocardial biopsies, in four patients we noted abnormal histology distinct from the usual type of congestive cardiomyopathy but with a strong resemblance to amyloidosis. The patients presented with unexplained ventricular tachycardia (N = 3) and/or congestive heart failure (N = 2). Biopsies revealed subendocardial, interstitial, and perivascular hyaline eosinophilic fibrillar material that did not stain with Congo red. Electron microscopy revealed that this material was organized into bundles of tangled microfibrils composed of twisted and tubular structures measuring up to 17 nm wide, which did not resemble amyloid or cyclosporin-associated microfibrils. Immunoelectron microscopy of the index case, using monoclonal antibody to fibrillin, specifically identified these structures as fibrillin microfibrils; fibronectin also was bound to the interstitial microfibrils. We believe that the subendocardial and interstitial deposition of microfibrils in these four symptomatic patients may represent a new type of infiltrative cardiomyopathy, similar to but distinct from cardiac amyloidosis. We do not know yet if this disorder is genetic or acquired, or if the prognosis is better than that of cardiac amyloidosis. However, atypical cases of primary cardiac amyloidosis should be reevaluated in light of these findings.

Intralysosomal lipid in long-term maintenance transplant atherosclerosis.

Intralysosomal accumulation of lipid has been implicated as an important mechanism in the pathogenesis of atherosclerosis. Although atherosclerosis develops frequently in organ transplants maintained on a long-term basis, to our knowledge no studies to date have demonstrated the intracellular localization of the lipid in this setting. Light and electron microscopic study of a renal artery branch from a transplanted kidney maintained for 3 1/2 years demonstrates that the lipid is sequestered within intimal smooth muscle cell lysosomes. The features of the atherosclerotic plaque in long-term transplantation appear to be identical to spontaneous lesions or those induced experimentally.