Simultaneous occurrence of a thyromediastinal muscle, a truncus bicaroticobrachialis, and a left superior vena cava.

A case is presented of a combination of anatomical anomalies found in a 67-year-old female cadaver during routine dissection by medical students. They include a thyromediastinal muscle, a truncus bicaroticobrachialis, and a left superior vena cava, with complete absence of the right superior vena cava, but with a normal azygos vein opening into the right atrium at the expected site of entry of the superior vena cava. No associated congenital cardiac malformations were found. Clinical implications include the difficulty of heart catheterization through the subclavian veins and misleading images on CT or MRI scans, where the azygos vein could be mistaken for a right superior vena cava.

Hepatic peroxisomes in isolated hyperpipecolic acidaemia: evidence supporting its classification as a single peroxisomal enzyme deficiency.

Hyperpipecolic acidaemia is still regarded as a peroxisomal assembly deficiency. The enzyme responsible for the accumulation of pipecolic acid is located in the peroxisomes in man. We studied the appearance and alterations of peroxisomes in liver biopsy material from three unrelated children suffering from isolated hyperpipecolic acidaemia, in which only the metabolism of pipecolic acid is disturbed, using light and electron microscopy after cytochemical staining for visualisation of peroxisomes. Morphometric results showed the presence of normal-sized to small peroxisomes, an increase in number and abnormally shaped organelles, suggesting enhancement of metabolic efficiency. In one case enlarged organelles were observed. Skin fibroblasts were studied in all patients: their peroxisomes appeared to be normal. The obvious presence of peroxisomes in isolated HPA indicates that this disorder should be classified as a single peroxisomal enzyme deficiency.

Biogenesis of peroxisomes in fetal liver.

Peroxisomes are single membrane-limited cell organelles that are involved in numerous metabolic functions. Peroxisomes do not contain DNA; the matrix and membrane proteins are encoded by the nuclear genome. It is assumed that new peroxisomes are formed by division of existing organelles. The present article gives an overview of microscopic studies and recent unpublished results dealing with peroxisome biogenesis in mammalian fetal liver and presents data on peroxisomes in oocytes. Cytochemical (catalase and D-aminoacid oxidase activity) and immunocytochemical data in rat and human liver (antigens of catalase, the three peroxisomal beta-oxidation enzymes, alanine: glyoxylate aminotransferase, peroxisomal membrane proteins with molecular weights of 42 and 70 kDa) indicate that during embryonic and fetal development the peroxisomal population undergoes a differentiation with respect to the composition of the matrix and to the size and number of the organelles. In the youngest stages, rare and small peroxisomes are present, into which the matrix components are imported in a sequential way. The import seems asynchronous in peroxisomes of the same hepatocyte. The size and number of the peroxisomes increase during liver development. In rat and human liver, no morphological or immunocytochemical evidence for an elaborate network of interconnected peroxisomes ("reticulum") was found. Instead, peroxisomes presented as individual organelles, which occasionally show membrane extensions. The importance of the metabolic functions of peroxisomes in human liver is emphasized by the peroxisomal disorders. In the liver of affected fetuses, the microscopic features associated with the defect can already be recognized; i.e., either catalase containing peroxisomes are absent and catalase is localized in the cytoplasm (in fetuses affected with Zellweger syndrome or with infantile Refsum disease) or peroxisomes are present but they are abnormally enlarged (e.g., a fetus affected with acyl-CoA oxidase deficiency). In the quail ovary, numerous peroxisomes are observed in the oocyte and in the granulosa cells during follicle maturation, but not in the full-grown egg. Thus, the mechanism of peroxisome inheritance remains unresolved.

Isolated dihydroxyacetonephosphate-acyl-transferase deficiency in rhizomelic chondrodysplasia punctata: clinical presentation, metabolic and histological findings.

UNLABELLED: Rhizomelic chondrodysplasia punctata (RCDP) is clinically characterized by symmetrical shortening of the proximal limbs, contractures of joints, a characteristic dysmorphic face, and cataracts. In the classical form an impairment of several peroxisomal functions and enzymes (plasmalogen synthesis, phytanic acid oxidation, 3-oxoacyl-CoA thiolase) has been repeatedly shown. Recently a variant involving only the peroxisomal dihydroxyacetonephosphate acyltransferase (DHAP-AT) has been described. We present a patient with isolated DHAP-AT deficiency and all clinical, radiological and pathological features of classical RCDP. For the first time, microscopy and immunocytochemistry of hepatocytes could be performed. CONCLUSION: In contrast to studies on classical rhizomelic chondrodysplasia punctata which have shown enlarged peroxisomes in numbers varying from hepatocyte to hepatocyte, the peroxisomes in our patient seem to be normal in size, number and shape.

Hyperoxaluria with hyperglycoluria not due to alanine:glyoxylate aminotransferase defect: a novel type of primary hyperoxaluria.

Considering the clinical heterogeneity of primary hyperoxaluria type I (PH1) and the fact that in many instances this diagnosis was made without enzymatic and immunohistochemical investigation, other disturbances of oxalate metabolism than those presently known can be expected in PH1. Using a gaschromatographic/mass spectrometric method that allows quantification of these acids, hyperoxaluria and hyperglycoluria was found repeatedly in two unrelated patients. The hyperoxaluria was unresponsive to pyridoxine. There was no nephrocalcinosis or urolithiasis. In the liver biopsy normal AGT activity and normal localization of this enzyme in the peroxisome was found. In one patient abnormal Km and maximal activity and mozaicism of AGT were excluded. Hyperoxaluria and hyperglycoluria were also found in other family members, suggesting autosomal dominant transmission. Although the underlying defect leading to hyperoxaluria and hyperglycoluria could not be identified in these patients, it is probable that they represent a separate type of primary hyperoxaluria.

Peroxisome mosaicism in the livers of peroxisomal deficiency patients.

Peroxisomal deficiency disorders, which are genetically transmitted, are assumed to be expressed in all cells, and the use of cultured skin fibroblasts for diagnosis and research is based on this assumption. We describe three patients with clinical, biochemical, and microscopic evidence of a peroxisomal disorder. However, their liver displays mosaicism, i.e., parenchymal cells with peroxisomes are adjacent to cells without peroxisomes. Ten percent (volume), 8%, and less than 1% of the parenchyma possessed peroxisomes that can be identified in immunocytochemical tests for six matrix and membrane proteins performed by light and electron microscopy. In the bulk of the parenchyma, catalase is localized in the cytoplasm, and in such cells no peroxisomes are evident by electron microscopy and immunolabeling for the 43-kd peroxisomal membrane protein (PMP) in two patients; in the third case, peroxisomal membrane ghosts are present. Immunoblots of peroxisomal beta-oxidation enzymes show a pattern similar to that from patients with a generalized peroxisomal deficiency. In contrast to the clinical and biochemical signs of peroxisomal dysfunction and hepatic histopathology, cultured fibroblasts from two patients demonstrate normal peroxisomal functions, including very-long-chain fatty acid oxidation and plasmalogen synthesis.

Practical guide for morphometry of human peroxisomes on electron micrographs.

Morphometry of peroxisomes is performed on electron micrographs of ultrathin sections after staining for catalase activity with diaminobenzidine; specific peroxisomal labelling is preferred to guarantee recognition. Peroxisomal number, size, axial ratio and volume parameters are determined and compared to control values. Results from 19 patients with loss of peroxisomal functions are listed. In many patients alterations in peroxisomal morphometric features are found. A brief guideline for interpreting morphometric data is included. Diagnostically relevant morphometric alterations are summarized.

Hepatocellular peroxisomes in human alcoholic and drug-induced hepatitis: a quantitative study.

The peroxisomes in the liver of four patients with alcoholic hepatitis and in six patients with drug-induced hepatitis are compared to eight control livers by catalase cytochemistry and morphometry. A decrease of catalase activity is observed in alcoholic, amitriptyline, aprindine, clomipramine and methiomazole hepatitis. Peroxisomes with a heterogeneous distribution of the catalase reaction product are found in most hepatitis livers. The number of organelles is increased 1.5 to 4.2 times in alcoholic, aprindine, methimazole and phenytoin hepatitis livers. In the last case, peroxisomes are also smaller. Changes in shape are seen in all hepatitis livers; they include invaginations, tails, funnel-like constrictions and gastruloid cisternae. In aprindine, phenytoin, methimazole and two alcoholic hepatitis livers, surface density exceeds the upper control value. These data indicate a loss of catalase activity in most hepatitis livers but also peroxisomal proliferation and shape modifications. It has been proposed that the latter changes are favorable for metabolic activity.

Fully automated measurements by light microscopy of tissue sections using a cellular array computer.

Software was developed for the acquisition, segmentation and analysis of microscopic OD-images on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board. The delineation algorithms for peroxisomes, lysosomes, and nuclei in liver, kidney, and adrenal gland sections start by thresholding the difference between the original image and a low pass filtered version. The resulting binary mask is then processed by morphological operations in order to produce an object overlay. The efficiency of the programs is evaluated by comparing delineated objects at different OD-levels, created by varying the stain or by multiplying the original pixel values with constant factors. Manual delineation on some images is also used as a reference. More complex algorithms are used for the delineation of muscle fibres in ATP-ase-stained sections and immunocytochemically labelled cells in monolayer preparations. Muscle images from parallel sections with different stainings are matched with a coordinate transform, enabling the transfer of the object mask from a single delineated image to the unprocessed images and thus obtain all necessary information for fibre classification. After segmentation, the OD-images and their object overlays are fed into a data extraction program, measuring for each delineated object user-selected features. Data are sent to a VAX for statistical interpretation.

Polarizing inclusions in some organs of children with congenital peroxisomal diseases (Zellweger's, Refsum's, chondrodysplasia punctata (rhizomelic form), X-linked adrenoleukodystrophy).

Polarizing material has been reported in the liver of children with infantile Refsum's disease (IRD) and was absent in two patients with the cerebro-hepato-renal syndrome of Zellweger (CHRS). We examined in polarized light 15 liver biopsy and autopsy samples from six other patients with the cerebro-hepato-renal syndrome of Zellweger, two with the rhizomelic form of chondrodysplasia punctata (rCDP) and two with X-linked adrenoleukodystrophy (ALD), all conditions with deficient peroxisomes. Two types of birefringent inclusions were found in CHRS only: the first is transparent in bright field, the second appears as brown granules or rods, similar to lipofuscins. As in IRD large PAS-positive macrophage-like cells contain the transparent type. Electron microscopical investigation of these cells shows trilaminar structures within membrane-bound organelles. The two types were also seen in kidney and brown adipose tissue, the first type in pancreas, the second type in adrenal gland; no such was observed in myocardium or in thyroid gland (CHRS). No birefringent inclusions were present in rCDP and ALD. The nature of the inclusions is still unclear. An accumulation of the transparent polarizing material with increasing age of the patients is most likely.

Myocardial H2O2 production in the unanaesthetized rat. Influence of fasting, myocardial load and inhibition of superoxide dismutase and monoamine oxidase.

Myocardial H2O2 production was studied by means of the in vivo administration of aminotriazole (AT), which inactivates the catalase-H2O2 complex compound I. Measurements of the residual catalase activity in male and female rats indicate that beta-oxidation of fatty acids in peroxisomes does not contribute in a substantial way to energy production in response to fasting or to an increased myocardial load, despite previous data on peroxisomes in myocardium. Superoxide dismutase (SOD) inhibition by diethyldithiocarbamate demonstrates that SOD participates in the production of H2O2 in physiological conditions. Such a role was not demonstrated for monoamine oxidase through inhibition by phenelzine.

Peroxisomal beta-oxidation from endogenous substrates. Demonstration through H2O2 production in the unanaesthetized mouse.

A system was developed in which it is possible to detect in vivo changes in hepatic H2O2 production, using a combination of the catalase inhibitor, 3-amino-1,2,4-triazole and methanol. In mice, starvation significantly increases hepatic H2O2 production and plasma non-esterified fatty acid concentrations. Short-term refeeding after a 24 h starvation period brings H2O2 production and plasma non-esterified fatty acid concentration back to normal in 3h. Administration of insulin 24 h after the onset of starvation normalizes H2O2 production in less than 2h and decreases non-esterified fatty acid concentration below normal values. The suppression by insulin of H2O2 production, as well as its coherence with plasma non-esterified fatty acid concentration, indicate that increased H2O2 production in starved mice reflects peroxisomal beta-oxidation.

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.