Max Wachstein

HISTOCHEMICAL DEMONSTRATION OF MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE ACTIVITY IN TISSUE SECTIONS

A comparative study was made of the distribution of mitochondrial adenosine triphosphatase activity in several organs of rat and rabbit using both the calcium and lead techniques. Certain modifications in these techniques assured both improved intracellular morphology and considerable preservation of enzyme activity. Of particular importance was the low temperature (–2 to –3°C) of the neutral calcium-formol solution, and, following short fixation (lead method), a subsequent wash in neutral buffer. Mitochondrial activity was similar with both techniques, but the lead method proved to be less costly, less time-consuming, and above all, far less capricious than the calcium technique. In good preparations, the appearance of mitochondria is as clearly defined as in sections stained with non-enzymatic, conventional techniques. Long exposure of cryostat sections to formalin or preparation of sections from tissue blocks fixed in neutral formalin leads to the complete abolition of mitochondrial activity; on the other hand, it accentuates enzyme staining of other structures, such as, for instance, bile canaliculi in the liver and the secretory capillaries in the pancreas and salivary glands. It also visualizes the infolding membranes in certain tubules of the rat and dog kidney. It is assumed that formalin-fixation aids in the enzymato-morphologic distinction of these two different intracellular structures.

DEMONSTRATION OF PEROXIDASE ACTIVITY IN TISSUE SECTIONS.

By using an improved benzidine technique, peroxidase activity can be demonstrated in various locations in mammalian tissues. A relatively formalin resistant enzyme is found in hemoglobin and is also associated with mitochondria of striated muscle and heart. A somewhat less formalin resistant peroxidase occurs in the granules of myeloid and mast cells. A relatively formalin sensitive peroxidase is present in a number of additional locations, e.g. the acinar cells in thyroid and salivary gland, the medulla of the kidney, in hair follicles of the guinea pig skin and Kupffer cells of the liver.

ADENOSINE TRIPHOSPHATASE ACTIVITY IN MELANOCYTES AND EPIDERMAL CELLS OF HUMAN SKIN

When crvostat sections of human skin are fixed at a low temperature (3#{176}C) for a short perio(l (15 minutes) in calcium-formol and processed to show adenosine tril)hosl)hatase activity by the lead method, three types of staining occur in the epidermis. a) Mitochondrial -SH dependent staining is seen in cyt.ol)lasmic structures of the epidermal cells. These are most numerous in the basal layer and their numbers decrease )rogressive1y in succeeding layers until none are found in the granular layer. Numerous mitochondria are seen in the defldriti(’ melanocvtes as well. b) The cell membranes of the dendritic melanocvtes are well outlined anti numerous (lark brown granules contained in these cells are stained also. These granules are assumed to correspond to melanosomes. c) Melanin granules, if present, in the epidermal (ells are stained darkly as well as the keratohyalin granules in the stratum granulosum. When crvostat sections are overfixed in formalin the staining in the mitochondria (a) is abolished. When crvostat sections are incubated in a medium without added adenosinetriphosphate mito(hondrial staining (a) as well as that of the (lendriti( melanocvtes and their contained granules (b) (hoes not occur. Ihe only structures stained under this condition are melanin granules in the basal epidermal cells and keratohyalin granules in the cells of the granular layer (c). If the sections are exposed to longer periods of formalin fixation prior to incubation in the ATP medium, mitochondrial staining (a) is suppressed, but non-mitochondrial specific and non-specific staining (h and c) are observed. I)endritic melanocytes are outlined by the activity of one enzyme only in this study, that of * Stipported in part by United States Public Health Grant A-688(C). t Presented, in part, at the Thirteenth Annual Meeting of the Histochemical Society at Atlantic City, New Jersey, April 12-13, 1962. non-mitochondrial, formalin-resi.stant adenosine triphosphatase. The activities of other Pho.sPhomofloesterascs in the human epidermis are compared with that of adenosine triphosphatase. In addition, the histochemical distribution of the activities of enzymes resumably localized totally (succinic dehydrogenase) or, perhaps, partially (diand tri-phosphopyridine nucleotide dial)horases) in the mitochondria are investigated anti coml)ared with the distribution i )attern for mitochondrial adenosine triphosphatase.

Pyridoxal phosphate in plasma and leukocytes of normal and pregnant subjects following B6 load tests.

Summary A method for determination of pyridoxal phosphate in human plasma is described. The amount of coenzymatically active form of Vit. B6 was determined simultaneously in blood of normal subjects, delivering women, and in cord blood of their babies. In general, amounts of pyridoxal phosphate in plasma and leukocytes showed parallel changes. They were low in maternal blood, and high in cord blood. Following oral administration of 100 mg pyridoxine hydrochloride, increases were observed in both plasma and leukocytic B6 values within a few hours. Considerably higher peaks were seen in normal controls as compared to women in last trimester of pregnancy and at time of delivery. The most significant difference between pregnant and nonpregnant subjects was noted in plasma values 24 hours following administration of vitamin. The possible practical application of these findings for evaluation of a latent B6 deficiency in clinical subjects is suggested.

Pyridoxal phosphate (B6-al-PO4) levels in organs, leukocytes and blood of rats with developing vit. B6 deficiency.

Summary Pyridoxal phosphate levels were determined in the heart, liver, kidney, muscle, circulating leukocytes, plasma and whole blood of rats on a synthetic diet with and without added Vit. B6. Concentration of B6-al-PO4 decreased in all tissues and in leukocytes, blood and plasma significantly within 1 to 2 weeks in animals on the deficient diet. The levels of pyridoxal phosphate diminished within 5 to 6 weeks to 10 to 15% of control values in all organs, circulating leukocytes, plasma and blood. It is concluded that measurement of pyridoxal phosphate in organs as well as in leukocytes, plasma and whole blood can serve as a sensitive indicator for assessment of the nutritional state of the animals in regard to Vit. B6.

Pyridoxal phosphate (B6-al-PO4) levels of circulating leukocytes in maternal and cord blood.

Summary Comparison of the pyridoxal phosphate content of circulating leukocytes in non-pregnant controls, women at term and umbilical cord blood shows significantly higher values in normal controls as compared to women at term. The cord blood shows the highest levels. Pyridoxine administration elevates the B6-al-PO4 levels in pregnant women to a very marked, and that of cord blood to a moderate, degree. It is concluded that the growing fetus can successfully compete with the mother for this important vitamin.

Pyridoxal Phosphate in Plasma and Leukocytes in Patients with Leukemia and Other Diseases.

Summary The amount of coenzymatically active Vit. B6 was determined in plasma and leukocytes in patients with various diseases. Values for plasma and leukocytes varied widely in all groups examined. A statistically significant decrease of plasma B6 was found only in patients with nutritional anemia and various forms of leukemia. However, the amount of pyridoxal phosphate was very low in individual patients with various other diseases. This may indicate a decreased intake of vitamin in some hospitalized patients or an increased need for it. B6 values in leukocytes were consistently decreased in patients with various forms of leukemia. In contrast, cells from normal bone marrow and from tonsils showed significantly higher amounts of pyridoxal phosphate. The findings are discussed with reference to a specific disturbance in B6 metabolism in some neoplastic conditions.

Intracellular Bile Canaliculi in the Rabbit Liver

Summary With the aid of the histochemical phosphatase stain, intracellular bile canaliculi can be demonstrated in the normal rabbit liver. These intracellular branches become considerably more pronounced following experimental biliary obstruction.

Occurrence of Hemorrhagic Centrolobular Liver Necrosis in Protein Deficient Rats.

Summary On a synthetic protein-free diet containing all other essential nutrients with or without a 2% casein supplement, the livers of young male Wistar strain rats show centro-lobular necrosis, often associated with hemorrhages in about 50% of animals. Incidence of these lesions decreases when casein content of diet is increased. Addition of 1% methionine is of no influence, while 0.5% cystine often aggravates the severity and incidence of centrolobular necrosis. The findings are discussed with reference to hemorrhagic centrolobular necrosis which occurs frequently after administration of ethionine, a supposed methionine antagonist.

Influence of God Liver Oil on Deposition of Ceroid in the Nutritional Rat Cirrhosis

Summary Regular and severe liver cirrhosis can be induced in male weaning rats on a choline-free, protein-deficient diet after 90 days, if the protein source is either fibrin or peanut meal plus casein. Early cirrhosis is occasionally observed in less than 60 days. Very large amounts of ceroid are deposited by the use of these diets, containing 2% cod liver oil. The amount of the deposited ceroid is markedly reduced if the intake of cod liver oil is restricted to one drop weekly. On the same diet without any cod liver oil, the produced cirrhosis is less severe. Some of these livers contain no ceroid, others small amounts of this pigment. Cod liver oil is therefore not the only cause for ceroid pigmentation in nutritional cirrhosis. It enhances, however, its formation very markedly.