Baker's Acid Hematein Research Articles

Localization of phospholipids in human catecholamine neurons with Baker's acid haematein

Protein bodies in catecholamine neurons of the normal human brain contain arginine-rich proteins similar to those present in the core of Lewy bodies in Parkinsonian brains. Lewy bodies are known also to contain phospholipids in their core, demonstrable with Baker's acid haematein. We used Baker's procedure on catecholamine neurons of normal brains to test whether the protein bodies also contain phospholipids. Postmortem tissues of three control individuals were used in this study. Locus coeruleus and substantia nigra were dissected out from the fresh brains and two sets of blocks were made from each area. One set was fixed in formol-calcium for the preservation of lipids, the second was fixed in Bouin's solution and treated with hot pyridine for the extraction of lipids. Finally, both sets were subjected to the same chroming and staining procedure according to Baker. In catecholamine neurons the protein bodies were stained with acid haematein, indicating the presence of phospholipids. Since this staining resisted pyridine extraction we conclude that these phospholipids are firmly bound to the basic proteins. Thus, protein bodies in healthy catecholamine neurons give the same positive reaction for phospholipids as that reported for the core of Lewy bodies in damaged neurons in Parkinsonism.

Cellular chemical indices of right ventricular protection in children.

Myocardial preservation during open heart surgery in children was studied using biopsies of the right ventricle taken at the beginning and end of a bypass with a Tru-Cut biopsy needle. Three assessments were used: (1) semi-quantitative cytochemical grading of Baker's acid haematein reaction, and the distribution of succinate dehydrogenase and myosin ATPase; (2) microdensitometry of the succinate dehydrogenase activity; and (3) quantitative birefringence measurements to assess the response of the myocardial fibres to ATP and calcium. For each assessment, the values at the beginning and end of the bypass were compared. In a series of 42 children, two died in low cardiac output and three others required substantial inotropic support. No patient showed deterioration in the overall cytochemical assessment. Two patients showed deterioration in birefringence, one died and one had low cardiac output. The remaining three patients who had post-operative problems had low birefringence values at the beginning of bypass. Thirty-two patients were used for the microdensitometric assays, one died and three required substantial inotropic support. Succinate dehydrogenase activity decreased significantly during bypass in only two patients. One of these died and the other required substantial inotropic support.

Histochemical localization of phospholipids in the olfactory epithelium of fish.

Histochemical localization of phospholipids in the olfactory epithelium of rainbow trout (Salmo gairdneri), whitefish (Coregonus clupeaformis), Arctic char (Salvelinus alpinus), brook trout (Salvelinus fontinalis), lake trout (Salvelinus namaycush), and black bullhead (Ictalurus melas) was examined. The present results indicate that phospholipids are highly localized in the receptor neurons of all species examined, present not only in the membranes, but also in the cytoplasm. Although the phospholipids of membranes and cytoplasm may not be of the same kind, their localization suggests a role in olfactory function. The Baker acid hematein method shows clear morphological features of the olfactory neurons beyond those obtainable by routine histological methods. The technique may be useful in determining morphological changes and (or) phospholipid alterations caused by a deleterious environment.

Lipidosislike renal changes in rats treated with chlorphentermine or with tricyclic antidepressants

Histological, histochemical and ultrastructural examinations were performed on renal tissues of rats after prolonged oral administration of the anorectic drug chlorphentermine or of the tricyclic antidepressants iprindole, imipramine and clomipramine. All drugs caused the formation of multilamellated cytoplasmic inclusions throughout the nephron and the collecting duct system, and in interstitial cells. The cytological alterations were most prominent in the glomerular podocytes, in the proximal convoluted tubules, and in the collecting duct system. In view of the histochemical properties (staining with Baker's acid hematein) and the ultrastructural appearance of the cytoplasmic inclusions the cellular alterations are interpreted as a renal manifestation of a generalized lipidosis induced by drugs of amphiphilic character.

The effect of postmortem trauma on neuronal cell types stained histochemically for phospholipids

With Baker's acid hematein method, blue and yellow neurons occur after fixation by immersion or after inadequate fixation by perfusion as the consequence of postmortem trauma. They increase in number when such trauma is intentionally applied to cerebral surfaces prior to fixation by either procedure, and their formation is prevented by successful perfusion of the fixative prior to removal of the cranium. The formation of blue and yellow cell types is not influenced by variations in composition of the fixative, embedding procedure or tissue preparation, age or species of animal, or treatment with pyridine. Prolonged delays in fixation do not affect the affinity for acid hematein in neurons next to superficial injuries caused by dissection. The two cell types are therefore regarded as artifactitious, comparable to dark neurons demonstrable by other staining techniques. It is concluded that the neuronal stainability with Baker's acid hematein is nonspecific and that the identification of neurons according to histochemical composition is equivocal in both normal and experimental material.

A comparison of the Baker acid Haematein staining of selected regions of rat hypothalamus with the fatty acid composition of their phosphatidyl choline content and their polar lipid fraction as determined by gas liquid chromatography.

Application of Baker's acid Haematein test for the histochemical detection of phospholipids to rat hypothalamus reveals striking colour differences between white and grey matter. It was found previously that these differences could not be attributed to corresponding differences in the acid Haematein-positive lipid content in various regions of the hypothalamus. As the staining reaction might depend on the degree of unsaturation of lipids, the fatty acid compositions of phosphatidyl choline and the polar lipid fraction from the optic chiasma and some regions of rat hypothalamus were investigated.

Phospholipid composition of some parts of rat hypothalamus, and its relation with some histochemical observations.

Abstract—In continuation of a histochemical investigation of phospholipids in rat brain, the content and composition of these lipids in some regions of rat hypothalamus were examined using ultra‐micromethods. The supraoptic and paraventricular nuclei, the anterior area of the hypothalamus, and the adjacent optic chiasma were chosen for this study. The phospholipid content of all of these regions was found to be almost equally high. Proportionally higher amounts were found for phosphatidylethanolamine in the optic chiasma and for phosphatidylcholine in the neurosecretory nuclei. In all regions investigated the sphingomyelin content was nearly the same. From these results it was concluded that the large differences in staining properties of myelinated and unmyelinated areas of the tissue when applying Baker's acid haematein test could not be ascribed to large differences in the content of the Baker‐positive, choline‐containing, phospholipids. Treatment of fixed tissue with cold acetone in order to extract non‐polar lipids that may mask phospholipids, did not abolish the staining differences.The possible involvement of cerebrosides and proteolipids mainly found in myelinated tissue in the Baker reaction is discussed. It is suggested that the differences in staining properties might be ascribed to differences in structure of myelinated and unmyelinated tissue, rather than to differences in the content of Baker‐positive lipids.

Application of standard micro-anatomical staining methods to epoxy resin-embedded sections.

Staining of semi-thin sections of osmium-fixed epoxy resin (Epon or Araldite)-embedded tissues with haematoxylin and acid dyes as counterstains (eosin or phloxin) was successfully achieved after intense oxidation using hydrogen peroxide as the oxygen donor. Results are closely similar to those obtained with paraffin sections (haematoxylin and eosin): nuclei stain blue, cytoplasm and extracellular structures (eg, collagen, elastica, basement membranes) stain with varying shades of red. Preliminary studies show that similarly good results, comparable with standard paraffinembedded histological materials, can be obtained, using H(2)O(2) pretreatment, with Weigert's iron haematoxylin, Mallory's phosphotungstic acid haematoxylin, Alcian blue, PAS reaction, Sudan III, Sudan black, oil red, Gomori's aldehyde fuchsin, and Baker's acid haematein.

The demonstration of phospholipids in certain cells in neurosecretory nuclei in the rat with Baker's acid Haematein after Fixation in glutaraldehyde-formol-calcium.

With Baker's acid haematein test certain ganglion cells in the brain, their processes and, at some sites, glial cells around blood vessels stain dark blue. This article describes a study of the Baker-positive cells which occur in and around the neurosecretory nuclei. By substituting formol-calcium fixation with glutaraldehyde-formol-calcium fixation shrinkage in brain tissue is completely avoided. If such fixation is used the argument that positive staining of ganglion cells with Baker's method only indicates that these are “shrunken neurons” can no longer be maintained. A comparative histological study, especially of Baker's technique and “controlled chromation” (Elftman) showed that the Baker-positive cells contain a phospholipid, probably bound to a protein, as a labile compound, which is easily lost. We found that to immobilize and localize this labile compound in the ganglion cells the technique of fixation and the pH during chromation (which should be around 3.8) are of fundamental importance. Only under these conditions is the complex sufficiently immobilized to allow of its demonstration with acid haematein. These requirements are now completely met if Baker's acid haematein technique is used. The article stresses that only prefixed and chromated frozen sections can be used for this method, thus avoiding shrinkage and non-specific staining of proteins. The modified Baker method as used by us gives constant and reproducible staining and is described in this article. The functional significance of the Baker-positive reaction in some ganglion cells in the n. s. nuclei or glial cells around blood vessels is not dealt with in this article.

Phospholipids of the sexual segment of the kidney of the Indian house lizard, Hemidactylus flaviviridis Ruppell

The Indian house lizard, Hemidactylus flaviviridis breeds seasonally (1). In a sexually mature male lizard, from October to May of the following year, the secondary and tertiary collecting tubules of the kidney are hypertrophied forming the sexual segment. The development of the sexual segment is synchronous with the testicular cycle and maximum development occurs during the period when the testes are spermatogenically active (1). The sexual segment is not developed in the females in any season of the year; it is completely regressed in males during sexual quiescence (June and July). Injection of 4.0 mg of testosterone propionate over two weeks stimulated the regressed sexual segment to hypertrophy in males during quiescence and also in females (2). The apical portions of the cells of the sexual segment are loaded with granular secretions which are stained with Baker's acid haematein and phosphomolybdic acid-stannous chloride, indicating the presence of choline - containing phospholipids (1). The composition of the phospholipids of the sexual segment, which is a unique feature of the lizards and snakes, is unknown. This preliminary communication describes the isolation and quantitative estimation of phospholipids present in the sexual segment of the Indian house lizard, Hemidactylus flaviviridis Ruppell.

The reaction of unsaturated fats with the acid hematein test.

The reaction of saturated and unsaturated oils and fatty acids was examined in vitro with the Baker's acid hematein test. It has been found that oils whose molecules contain fatty acid components of two or more double bonds give a positive reaction with the acid hematein technique. The intensity of the reaction runs parallel with the number of double bonds.

ENZYMIC HISTOCHEMISTRY OF GRANULAR COMPONENTS IN DIGESTIVE GLAND CELLS OF THE ROMAN SNAIL, HELIX POMATIA

1. Cytochemical visualization methods for activity of acid phosphatase, β-glucuronidase, aminopeptidase and non-specific and E-600-resistant esterases were applied to digestive gland tissue from starved and feeding Helix pomatia. Other cytochemical methods used included Baker's acid hematein for phospholipid and the periodic acid Schiff method. 2. Calcareous cells stained only for acid phosphatase activity, by both lead-salt and an azo dye method. Calcium granules within these cells did not stain with the azo dye method while false-positive reactions in the granules were always obtained with the lead-salt method. Some increase in enzymic activity was detected in the cytoplasm of feeding animals. Secretory-resorption (SR) cells showed little activity for acid phosphatase. 3. The yellow granules in SR cells stained for β-glucuronidase activity in both starved and feeding animals. After feeding, SR cells showed an increase in enzymic activity, both in granules and more diffusely in the cytoplasm. Although some aminopeptidase activity was present, insignificant differences in location and intensity of the enzyme were detected. 4. SR cells stained intensely for non-specific esterase activity. Cytoplasmic staining for this class of enzymes was intense in feeding animals. Treatment with the inhibitor E-600 resulted in loss of cytoplasmic staining; activity persisted in yellow granules in both starved and feeding animals. Some activity was detected in small, colorless granules. 5. Phospholipid was detected in peripheral regions of the SR cells from starved and feeding animals. The reaction was diffuse in starved animals but concentrated at the luminal border of the SR cells in fed animals. Yellow granules of SR cells were periodic acid Schiff-positive. Especially in fed animals, periodic acid Schiff granules appeared at the luminal border of SR cells. Starved animals fed on lettuce leaves impregnated with horse-radish peroxidase showed accumulation of enzyme in vacuoles closely associated with the yellow granules. 6. The observations extend the concepts, advanced by earlier workers, that granular components of the secretory-resorption cells play a significant role in digestion in the snail. The location of activity of the several hydrolases studied, and the alterations in response to feeding, suggest that these classes of enzymes and the granules form a functioning unit in the physiology of intracellular digestion in Helix.

Adrenocortical histochemistry of intact and hypophysectomized goldfish

The consistent negative reports dealing with teleost adrenocortical histochemistry were considered to be at variance with the reported physiological activity of this tissue. The histochemical problem was reinvestigated utilizing a total of one hundred twenty-one completely xanthic goldfish ( Carassius auratus L.), of which thirty-five were hypophysectomized. The lipids were visualized by Sudan black B, Nile blue, Oil red O, Baker's acid hematein, and osmium tetroxide; glycogen by carmine and periodic acid-Schiff techniques; ascorbic acid by the acidified silver nitrate techniques. Each histochemical technique and control was first adapted to teleostean tissue by the use of material from intact goldfish. The adrenocortical tissue of intact goldfish showed sudanophilia, osmiophilia, neutral lipids, phospholipids, cholesterol, glycogen, and ascorbic acid. The adrenocortical tissue of the hypophysectomized fish, when compared with normal fish tissue, showed decreased sudanophilia, osmiophilia and phospholipids. Cholesterol and neutral lipids were barely demonstrable, while no detectable gross change occurred in the glycogen and ascorbic acid content of the cells two weeks after hypophysectomy. The findings of the present study indicate that the histochemical reactions of the goldfish adrenocortical tissue are comparable to those of the zona fasciculata and zona reticularis of the mammalian adrenal cortex. The previously reported negative histochemical findings in the case of the teleost adrenal cortical tissue may be ascribed to the failure to adapt the histochemical techniques to teleostean tissues and to account for alterations resulting from the “stress” of the experimental fish.

Evidence for lipid material in chromosomes

1. 1. A histochemical investigation has been made of a differential staining behaviour observed in chromosomes when sections of roots are stained in a mixture of orange G-aniline blue. 2. 2. The staining behaviour is dependent on the type of fixation. After Lewitsky's fixative the chromosomes stain uniformly yellow from about midprophase until the end of anaphase, but blue during telophase and interphase except in heterochromatic regions which stain yellow. After some other fixatives, e.g. acetic alcohol or formal-calcium, the chromosomes stain uniformly blue at all times. The nucleoli on the other hand stain yellow after these and most other fixatives. 3. 3. A closely parallel staining behaviour was found when sections of roots were treated with Baker's acid haematein test for phospholipids, indicating that the yellow staining of the chromosomes with orange G-aniline blue was associated with the presence of phospholipid. 4. 4. Material shown to be lipid has been recovered from formal-calcium solution used for the fixation of root tips but not from similarly used Lewitsky's fluid. Smears of this material, when fixed in Lewitsky's fluid, stain with Sudan black, give a positive acid haematein reaction and stain yellow with orange G-aniline blue. Arginine was also found in the smears as shown by the Sakaguchi test. 5. 5. Biochemical confirmation of the presence of phospholipid in used formal-calcium solution was provided by means of partition chromatography which indicated that a substance which yielded α-glycerophosphate on hydrolysis was removed from the root cells by this fixative. 6. 6. The staining behaviour of the nucleoli in relation to that of the chromosomes, and some problems connected with the acid haematein test and other lipid stains are discussed briefly.

Studies on some properties of neurosecretory substance. I.

1. In order to investigate lipid substances contained in the neurosecretory system in frog, Baker's acid hematein test in combination with the pyridine extraction method and Lison's sudan black technique were employed. Phospholipid was demonstrated in the preoptic nucleus of the hypothalamus and in the posterior lobe of the hypophysis. The distribution of this substance corresponded with that of Gomori's neurosecretory substance. Non-phospholipid material was found in the middle lobe, while, the phloxinophile substance by means of Gomori's stain was demonstrable, which was thought to be different from the neurosecretory substance in the posterior lobe. 2. Changes in stainability of the neurosecretory substance of rat pretreated by serum were observed. Five μ frontal sections were prepared from the hypothalamus, with hypophysis attached, of rat fixed by Zenker-formol, Bouin-, susa fluid or 10% formalin. These sections were incubated in a serum fluid of rabbit, following by staining with Gomori's C.H.P. stain. The stainability of neurosecretory substance was lost or decreased after being treated with the serum. When enzyme inhibitors, such as citric acid, cupric sulfate, lead acetate, mercuric acetate or formalin are added to the serum, the neurosecretory substance is easily stained as well as that of the control. It is assumed that the neurosecretory substance is denatured by some proteolytic enzymes contained in the serum.

CYTOLOGICAL EVIDENCE FOR A ROLE OF THE CORPUSCLES OF STANNIUS IN THE OSMOREGULATION OF TELEOSTS

1. The effects of DCA, pitressin, water, and sodium and potassium chloride on the cytology of the corpuscles of Stannius were studied by means of Baker's acid hematein stain for phospholipids. The fresh water characin Astyanax mexicanus was used.2. Two kinds of changes in the cells of the corpuscles were brought about by the experimental procedures: degranulation, loss of cytoplasm and consequent decrease in size of cells accompanied the administration of potassium chloride, undiluted pitressin and long continued immersion in 1% sodium chloride, and hypertrophy of cells with increase in numbers and size of blackened granules accompanied the administration of water, diluted pitressin and DCA.3. Loss of staining reaction in mitochondria of kidney tubules was associated with increased secretory activity in the corpuscles of Stannius except in the case of long continued DCA administration and administration of pitressin.4. The results are interpreted as indicating a function of the corpuscles of Stannius in the osmoregulation of these fish possibly connected with excretion of excess water.

The Effects of Single Doses of Pilocarpine and of Histamine upon the Granules of Oxyntic Cells of Rats, with Special Reference to their Phospholipine Content

ABSTRACT New data are presented on the morphology and cytochemistry of the granules of the oxyntic cells of the rat’s stomach in continuation of work previously reported (Menzies, 1949). The effects of single injections of pilocarpine and of histamine on the granules were investigated, with particular reference to their phospholipine content as shown by Baker’s acid haematein method. Pilocarpine causes a marked enlargement of the granules with loss of phospholipine from the central part of many of the larger granules, and complete loss of phospholipine from others. The granules that have lost their phospholipine probably still contain lipoid as shown by sudan black. Pilocarpine also causes marked aggregation of the granules. Histamine causes a less marked increase in granule size and no observable loss of phospholipine, but some of the granules became elongated, an effect that can be seen with the phase-contrast microscope, and there is a tendency for them to recede to the peripheral parts of the cells associated with an enlargement of the intracellular canals. Both pilocarpine and histamine result in some enlargement of the oxyntic cells, the effect being more marked with pilocarpine. Neither pilocarpine nor histamine alters the size or shape of the non-lipoid moiety of the granules. Supravital colouring with neutral red shows a few droplets in oxyntic cells in the bases of the gastric glands. These enlarge slightly after histamine and greatly after pilocarpine, and they are always spherical. It has not yet been possible to decide as to their exact nature.

Cytochemical Reactions of Bovine Spermatozoa and Seminal Plasma

Summary By using accepted cytochemical techniques the following reactions were observed in bovine spermatozoa: Acid phosphatase was present throughout with a slight reaction in the equatorial segment when sodium glycerophosphate was used as a substrate at pH 5.0. Alkaline phosphatase was present, with the exception of the neck, when the same substrate was employed at pH 9.0. Enzymatic activity was found in the neck, middle-piece and tail when adenosine triphosphate was used at pH 5.0. The occurrence of an enzyme acting upon hexose diphosphate (fructose-l,6-diphosphate) at pH 9.0 was noted in the head, neck, middle-piece and tail. Gram's stain and the Feulgen reaction were positive throughout the head, indicating the presence of both ribonucleic and desoxy-ribonucleic acids. Metachromatic staining was not obtained with toluidin blue, indicating the absence of mucopolysaccarides. Fat, including phospholipid, was localized in the middle-piece. The acrosome was characterized by a slight positive reaction with the periodic acid-Schiff reaction which was not due to glycogen, glycolipid or mucopolysaccharide. Furthermore, the protoplasmic droplet which occasionally was present on the tail gave an intense reaction with the periodic acid-Schiff procedure. Seminal plasma gave the following reactions: Acid phosphatase was positive with sodium glycerophosphate as a substrate at pH 5.0 and alkaline phosphatase was characterized by a weak response with hexose diphosphate (fructose-1,6-diphosphate) at pH 9.0. Gram's stain and toluidin blue gave no reactions. Baker's acid hematein test for phospholipid was negative, but the control preparations were characterized by a bluish-grey stippling and a positive test was obtained with Sudan black B. No reaction was observed with Bodian's protargol method. A slight positive reaction was obtained with the periodic acid-Schiff reaction.

Observations on the Chemical Composition of Myelin and the Smallest Size of Myelinated Nerve-Fibres in the Central Nervous System

Baker's (1946) acid haematein and pyridine-extraction control tests, claimed to be specific for phospholipines (Baker, 1947), have been applied to various parts of the central nervous system of rats and man. The sudan black method for the detection of lipoids and the nile blue method for the staining of acidic lipoids have also been used. The findings are in agreement with older statements in the literature that myelin contains a considerable amount of phospholipines. It was impossible to determine whether galactolipines or neutral lipoids are also present. In the acid haematein-stained sections finer fibres were seen than when other stains for myelin sheaths are employed. Fibres with a diameter of 0.5 µ or even somewhat less were stained in various parts of the central nervous system of rats. It is regarded as probable from these findings that fibres down to 0.5 µ or even smaller possess a lipoid investment. These observations lend support to the now commonly accepted view that the distinction between myelinated and so-called unmyelinated fibres is arbitrary. Some observations are made, however, which indicate that the presence of truly unmyelinated fibres cannot be excluded.

An Examination of Baker's Acid Haematein Test for Phospholipines

1. Baker's acid haematein test for phospholipines is specific provided that only a definite positively result is considered. Very pale blues and greys may be caused by other lipoids, which if present in very large masses may possibly show medium to dark blue granules but will not be coloured all through. 2. The mechanism of the test appears to be as follows: (a) Phospholipine is not fixed by formal-calcium but is restrained from passing into solution by the calcium ions, which play no other part. (b) Phospholipine combines readily with chromium from the mordanting fluid, and is then rendered insoluble and mordanted. Other substances, acidic and usually containing phosphorus, are mordanted as well. (c) On staining, blue and brown colorations are formed; in both cases the dye attaches itself to the chromium in the various substrates. (d) On differentiation, some browns and most blues, particularly those with phosphoric substrates, remain nearly fast, but most browns and the weak blues of certain lipoids (not phospholipines) are greatly reduced or removed entirely. The period of differentiation must not be shortened. (e) Blue-staining lipoids (phospholipines) are distinguished from other blue-staining substances by an extraction with the lipoid solvent pyridine, after special fixation. The other substances, and any bound lipoid not removable with pyridine, remain. 3. Since the specificity of the test depends on the relatively greater affinity of phospholipines among lipoids for the mordant, the period of chroming must not be lengthened. 4. One reason why some substances are coloured after pyridine extraction but not after acid haematein is that in the former case they are precipitated and so concentrated; in the latter they are not. This is not a general explanation for the whole class of such substances.