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Pseudoisocyanin Staining of Insulin and Specificity of Empirical Islet Cell Stains
Abstract
Two closely related pseudoisocyanins, N,N'-diethyl-6,6'-dichlorpseudoisocyanin chloride and N, N'-diethylpseudoisocyanin chloride, were tested for their metachromatic staining behavior with oxidized insulin. N,N'-diethyl-6,6-dichlorpseudoisocyanin chloride gave nonspecific metachromasia with collagen, mucus, and mast cells of adult tissues; almost all tissues of rat embryos exhibited nonspecific staining. Nonspecific reactions were rarely observed in adult or fetal tissues with the extremely labile metachromasia of N, N'-diethylpseudoiso-cyanin chloride. When oxidation time and temperatures are carefully controlled, this reagent apears to be highly specific for insulin-containing cells and can be used as a selective stain for beta cells. Paraffin sections of formalin fixed material were oxidized 45 sec at 28-29 C in freshly prepared acidified permanganic (2.5% KMnO4, 1; 5% H2SO4, 1; distilled water, 7—parts by volume), decolorized 30 sec in 5% oxalic acid, and washed 5 min in running tap water. After rinsing in 2 changes of distilled water, sections were stained 20 min in a 36 mg/100 ml aqueous solution of N, N'-diethylpseudoisocyanin chloride. Sections were then washed in running tap water until the albumen adhesive was decolorized, and mounted in Karo syrup diluted with an equal amount of distilled water. The insulin-containing cells are stained light to dark purple; all other tissue components, various shades of red. N, N'-diethylpseudoisocyanin chloride was used as a reference for evaluating the specificity of 5 commonly used empirical methods for demonstrating alpha and beta cells in pancreatic islets. Cells exhibiting pseudo isocyanin metachromasia were stained selectively by aldehyde-fuchsin, Heidenhain's azan, and chrome-hematoxylin. Aldehyde-Iuchsin was the only empirical stain tested which gave results comparable to pseudoisocyanin for clarity and definition of beta cells. After oxidation in acidified permanganate, azocarmine and phosphotungstic acid-hematoxylin differentially stained alpha cells; cells demonstrated by these two methods did not exhibit pseudoisocyanin metachromasia. This histochemical procedure can precede empirical methods which require preliminary oxidation in acidified permanganate or it can follow empirical methods which do not extract the insulin nor alter its intramolecular disulfide bonds.
... An alternate histochemical technique for staining islet B cells, based on the metachromatic reaction of oxidized insulin with pseudoisocyanin, was described by Coalson (1966). In this protocol, the three cysteine disulfide bonds of insulin are oxidized to sulfonic acid groups, which are subsequently detected with pseudoisocyanin dyes, using a method published by Schiebler and Schiessler (1959). ...
... Indeed, as late as 1966, Coalson stated in reference to the Lacy and Davies fluorescent antibody technique for identifying B cells, "…. fluorescent antibody procedures are too complicated for routine use by many laboratories" (Coalson 1966). ...
Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes.
© The Author(s) 2015.
... Fixed tissue paraffin 4-5 µm sections of pancreas were used for histochemistry of insulin and Zinc in B-cells using of methods as aldehyde fuchsine [7], Diethylpseudoisocyanine chloride [8], [9], [10], [11], [12], [13], [14], [15] Victoria Blue 4R [9], or with anti-insulin antibody using the indirect immunoperoxida-se method [10]. The fluorochrome 8-para(toluenesulphonylamino)quinoline (8PTSQ) as well as Dithi zone for staining and to analyze Z n2+ distribution in the pancreatic islet's sections [13]. ...
BACKGROUND: Amount of zinc-ions is correspond to insulin content in cytoplasm of β-cells. Diabetogenic zinc binding chemicals (DZC) formed with zinc in β-cells chelat complex that result destruction and death of β-cells within 10–15 min in animals. AIM: The aim of the study was to investigate using of specific histochemical methods the content of zinc and insulin in β-cells of pancreatic islets of human intact fetal pancreas tissue and under action of diabetogenic and non-diabetogenic zinc-binding substances diabetogenic zinc diethyldithiocarbamate (DZ and DDC). METHODS: Pancreas from 8-week-old human embryos and adult rabbits were used. Fixed or frozen islets were used in vitro effect. Histochemical methods for insulin and zinc in β-cells were used. RESULTS: Results showed that zinc in human β-cells is clearly revealed using of histochemical methods. In embryo pancreas, not integral islets were found as clusters of cells or even individual β-cells. Insulin content in ß-cells is for 7–20% lower and Zn2+ by 16–18% in compared with rabbits; Zinc in human ß-cells formed chelat complexes with 8PTSQ and DZ as Zn2+ –8PTSQ and Zn2+–DZ. Sodium DDC, a non-diabetogenic Zn2+ chelator, formed not toxic complexes with Zn2+ in fetal β-cells that protect cells of destruction caused by DZC as in animal’s pancreas. CONCLUSION: (1) Intracellular reactive zinc contained in human fetal pancreatic ß-cells clearly revealed using of histochemical methods and also (2) demonstrated that DZC in human ß-cells result formation with zinc of chelat complexes. (3) As in animals, non-toxic chemical as DDC is able to block zinc in ß-cells that result prevention of its destruction caused by DZC.
... Immunohistochemical (IHC) techniques were first described in 1942 as a method of detecting pneumococcal antigens using chemically-labeled antibodies (5). The use of IHC to identify pancreatic endocrine cells followed shortly after (6)(7)(8), though the IHC technique was not immediately widely adopted due to a lack of access to experimental tools such as antibody development and fluorescence microscopes (9). The development of IHC techniques allows for more specific and reproducible islet imaging as well as providing means of cell identification based on endocrine hormone production (e.g. ...
The pancreas is regarded as consisting of two separate organ systems, the endocrine and exocrine pancreas. While treatment of a disease with either an endocrine or exocrine pathogenesis may affect the function of the entire pancreas, the pancreatic diseases have been treated by clinicians in different medical disciplines, including endocrinologists and gastroenterologists. Islet microcirculation has long been considered to be regulated independently from that of the exocrine pancreas. A new model proposes that pancreatic islet blood flow is integrated with the surrounding exocrine capillary network. This recent model may provide revived or contrasting hypotheses to test, since the pancreatic microcirculation has critical implications for the regulation of islet hormones as well as acinar pancreas functions. In this mini-review, practical applications of in vivo and in situ studies of islet microcirculation are described with a specific emphasis on large-scale data analysis to ensure sufficient sample size accounting for known islet heterogeneity. For in vivo small animal studies, intravital microscopy based on two-photon excitation microscopes is a powerful tool that enables capturing the flow direction and speed of individual fluorescent-labeled red blood cells. Complementarily, for structural analysis of blood vessels, the recent technical advancements of confocal microscopy and tissue clearing have enabled us to image the three-dimensional network structure in thick tissue slices.
... High specific for Zinc fluorescent method of staining by 8PTSQ (8-para (toluenesulhonylamino) quinolin[2][3][4][5][6][7][8][9][10].Recommendations for Procedure of Embedding of PI inParaffin: a) Liquid paraffin is poured into a plastic tube (diameter 1 cm, height -2 cm) in a water bath at 56 0 C; raising of syringe up to the level of 0.5 cm from the top of the tube; d) Then remove the tube from the water bath. Thanks to this Technology: 1) Islets are evenly located over the entire height of the paraffin block; 2) It is possible maximally prevent formation of air bubbles in the paraffin near the islets Reagents Collagenase (Boehringer Mannheim GmbH, Germany); Diethylpseudoisocyanine (SERVA, Germany); Aldehyde-fuchsine (MERCK, Germany;) kits from DAKO for immunohistochemistry of insulin; 8PTSQ (Institute of High Pure Reagents, Moskva, Russia) Method of staining of insulin in β-cells of PI by Aldehydefuchsine histochemical technic our modification [pancreatic islet. ...
... High specific for Zinc fluorescent method of staining by 8PTSQ (8-para (toluenesulhonylamino) quinolin[2][3][4][5][6][7][8][9][10].Recommendations for Procedure of Embedding of PI inParaffin: a) Liquid paraffin is poured into a plastic tube (diameter 1 cm, height -2 cm) in a water bath at 56 0 C; raising of syringe up to the level of 0.5 cm from the top of the tube; d) Then remove the tube from the water bath. Thanks to this Technology: 1) Islets are evenly located over the entire height of the paraffin block; 2) It is possible maximally prevent formation of air bubbles in the paraffin near the islets Reagents Collagenase (Boehringer Mannheim GmbH, Germany); Diethylpseudoisocyanine (SERVA, Germany); Aldehyde-fuchsine (MERCK, Germany;) kits from DAKO for immunohistochemistry of insulin; 8PTSQ (Institute of High Pure Reagents, Moskva, Russia) Method of staining of insulin in β-cells of PI by Aldehydefuchsine histochemical technic our modification [pancreatic islet. ...
... Schiebler T. and Schiessler S. showed that A chair of oxidized insulin reacted with Diethylpseudoisocyanine chloride with formation of red fluorescent complex which fluoresces in UV light 360-370 nm. We have used modernized method [4,5]. Staining procedures. ...
... The positive metachromatic reaction obtained with pseudo-isocyanin also strongly indicates the presence of insulin. 28 Definite confirmation of the presence of insulin in the subcutaneous tumor cells was obtained by means of immunoassay and the fluorescent antibody technic. Similarly, the weak reaction for tryptophan is compatible with the presence of small amounts cME^glucagon, but does not prove it. ...
This paper describes some histologic and histochemical characteristics of an easily transplantable islet-cell tumor of the Syrian hamster. The presence of insulin was demon-strated by means of fluorescent antibody and immunoassay technics. A positive histochemical reaction for tryptophan suggests, but does not prove, the presence of small amounts of glucagon. It is suggested that the tumor may be a use-ful tool for biologic studies. DIABETES 16:409-14, June, 1967.
It is known that zinc possesses important role in processes of synthesis and formation of the deposited form of in pancreatic B-cells. Pancreas of many animals as of human contains a large amount of zinc. It is known also that some of chemicals and drugs capable to interact with zinc in B-cells that result destruction and death of cells. Authors have investigated the content of zinc inpancreatic islets of rabbit and features of localization in B-cells. It is established that the main amount of zinc concentrates in B-cells adjoining walls of blood capillaries through which there is hormone exit in blood. Authors suppose that destruction of pancreatic islets past intravenous injection of diabetogenic chemicals result for the first of all — destruction of B-cells adjoining to blood capillaries in islets.
Authors investigated a possibility of prevention developing of experimental diabetes in animals caused by group diabetogenic Zn-binding substances by preliminary injection of amino acid L-Hystidine contains the sulfhydril SH-groups in structure of molecule. It is established that intravenous administration of solution of 900-1000 mg/kg L-Hystidine to rabbits leads to the almostcomplete binding of ions of Zinc in B-cells of pancreas for 24 hours and prevent of formation in B-cells of a toxic complex Zinc-dithizon that is followed by prevention of developing of diabetes at 11 animals from 12. At the same time by comparative using of various histochemical research technics staining of tissue of pancreas it was shown that most sensitive for identification of insulin and Zinc in B-cells are fluorescent methods. The most qualitative results of assessment of features of gistotopography of insulin and Zinc in B-cells obtained by using of Aldehyde-fucshine method.
Работа посвящена исследованию возможностей использования в экспериментально-клинической практике трансплантационного метода лечения сахарного диабета. Показано, что в перспективе этотметод, в отличие от инсулинотерапии, несомненно, может стать методом излечения. Подчеркнуто, что особое значение имеет получение достаточного количества материала для трансплантации в виде изо-лированных и выделенных из поджелудочной железы панкреатических островков, качественная очистка их от примесей и подготовка к трансплантации путем культивирования с использованием ростовых факторов. Показана важная роль визуального гистологического контроля каждой партии полу-чаемых островков; авторами приведены собственные результаты гистологического и гистохимического контроля полученного материала с помощью модифицированных ими всех известных на сегодня вмире основных высокоспецифичных методов исследования состояния гистоструктуры и оценки содержания инсулина в В-клетках. На основании собственного значительного многолетнего опыта работы в западноевропейских лабораториях авторами предложены полезные замечания по вопросам организационного характера при проведении исследований в этом направлении.
Метод культуры изолированных панкреатических островков(ОПЖ) имеет два решающих преимущества по сравнению с опытами на животных, а именно: 1) можно исследовать характер прямого влияния исследуемых веществ на клетки тканей; 2) имеется возможность изучать влияние точно заданных концентраций исследуемых веществ на клетки исследуемой ткани. В работе представлены результаты применения изолированных ОПЖ для изучения характера прямого воздействия изучаемых веществ на β-клетки с помощью нескольких гистологических и гистохимических методик, дополняющих друг друга. Авторами показано, что каждый из нескольких методов обладает определенными очевидными преимуществами перед другими относительно возможностей оценки состояния гистоструктуры ОПЖ, чувствительности метода и содержания инсулина в β-клетках. Выявлены также преимущества технического характера отдельных гистохимических методов при работе с ОПЖ. При получении, очистке и отборе ОПЖ авторами на основании своего опыта установлено, что окончательный отбор материала лучше производить мануально, путем ручного отбора островков при визуальном микроскопическом контроле. В процессе заливки ОПЖ в парафиновые блоки авторы предлагают технологию заливки, обеспечивающую равномерное распределение ОПЖ в процессе заливки в парафиновом блоке по его вертикали, предупреждающую их оседание на дно в виде одного-двух слоев.
Authors investigated the nature of the direct effect of the diabetogenic derivatives of 8-hydroxyquinoline (TSQ) on pancreatic β-cells in experiments on a tissue culture model of isolated pancreatic islets (IPI), which allows us to study the direct effect of target concentrations of investigated substance on cells, whereas in experiments on animals, the metabolism of a substance injected into the body with the subsequent effect of metabolites on β-cells is not excluded. In experiments with IPI of newborn rats, TSQ was added to the nutrient fluid; after cultivation, the material was fixed in Bouin liquid. Histological and highly specific histochemical methods specific for insulin and zinc in β-cells were used to assess the insulin content in β-cells and the state of the islet’s histostructure. Results. It was shown that the direct effect of TSQ on β-cells is accompanied by cell destruction and a significant decrease of insulin and zinc content (from 1.68–1.74 pu in intact islets to 1.04–1.18 pu in experience), which is the reason for the destruction of islets and the development of type 1 diabetes. When choosing research methods, the most complete results are obtained by using of a complex that includes two methods — histological or combining the properties of histological and histochemical and one of the histochemical specific method for insulin and zinc in β-cells. In manual selection of purified pancreatic islets, the number of damaged IPIs is several times less compared to the other methods.
It is presented a developed and constructed histophotometric complex for quantitative evaluation in relative units of content of deposited insulin and zinc in insulin-producing B-cells of histologic preparations of pancreas. The method previously used in similar studies was based on a visual assessment of the results of coloring the preparations, or a more accurate quantitative one under the conditions of using universal complexes, including a microscope with built-in measuring devices (photometers), the disadvantage of which is, as a rule, a very high cost limiting the possibility of their use. In addition, such complexes, in connection with their universality, are not suitable for studies related to the study of the functional state of pancreatic B cells using highly specific histochemical methods for detecting insulin. The authors aimed to develop and create a studyoriented histophotometric complex of zinc and insulin in B cells, created using inexpensive components, which would help make quantitative analysis significantly more accessible to researchers working in the field of experimental diabetology.
This chapter discusses the histoenzymology of the islets of langerhans in poikilotherms, birds, and mammals. The existence of the islets of langerhans is known from the ammocoete larva to the most evolved teleost. The islets in cyclostomes have only one type of cell. The vacuolise under the influence of glucose injections and cauterisation causes hyperglycaemia, so it can be deduced that the islets are insulinogenic. Recent work has defined the very primitive nature of the islets of langerhans in cyclostomes. The cytochrome oxidase activity of the islets in Opsanus tau is quite strong, reaching 18% of the activity of the heart. Homogenates of the corpuscles of this species has similar hexokinase reaction. The variations in blood sugar that accompany the variations in islet cytology in reptiles are of particular slowness and amplitude. It is worth noting that cells of the same organ receiving the same blood, often adjacent to one another as are α and β islet cells, can elaborate two such different secretions—the one causing hypoglycaemia and the other hyperglycaemia.
Long-term administration of pyridoxine to rats kept on a diabetogenic diet stimulating endogenous synthesis of xanthurenic acid resulted in minimal glycemia, less pronounced decrease in insulin content in β-cells, and more intensive excretion of xanthurenic acid with urine. Histological changes were observed in 23% pancreatic islets, whereas in rats not treated with pyridoxine, destruction and necrosis of 40-45% β-cells were found in 38% of studied islets.
In the paper a technique of the polarization optical qualitative and quantitative detection of polyanions with N,N'-diethylpseudoisocyaninchloride in the histological section is described. It is possible to prevent depolymerisation after precipitation with potassium ferricyanide and to intensify the birefringence of the histological structures.
In vitro-attempts and measurements of the dispersion of the double refraction and of the dichroism indicate connections between the optical appearances, forthermore it is possible to make statements about the mechanism of the arrangement of the tissue-dye-ferricyanide-complex.
The efficiency of the practised methods for the visualisation of the cell types in the islets of Langerhans is now being investigated. We may divide these methods into the following groups: 1. Histological dye, 2. Silver impregnations, 3. Histotopochemical reactions, and 4. Electron-microscopic investigations.
Only those histological techniques are used which can demonstrate indubitable the A and B cells. The histotopochemical possibilities for the representation of islet cells (especially immunohistochemical techniques), are evaluated to find their essential importance for islet research. It is to be emphasized that this hormone detection method has provided us with a new picture of the performance of islet-cells in mammals.
Electron-microscopy has its role to play here, too, for electron-microscopy enabled first the confirmation, then the substantiation of the now classical conception of the 3 islet cell types. However, electron-microscopy has so far only been able to contribute little to our knowledge of the multiplicity of hormones which can be formed in these cells. Other methods will be able to elucidate this matter in the future, for example correlative light- and electron-microscopy, semithin microtomy, ultra-histochemistry, direct investigation of hydrous objects, and other new, or the latest methods in electron-microscopy.
Silver impregnation methods are being investigated for their importance for explaining nesocytology, in the past, as well as for their importance for present day islet research. We would like to draw attention to the often used possibilities for investigating. tissue dealt with in this manner under an electron-microscope.
Further, we would like to point out that connections between islet research and diabetology exist. For this reason the question of an exact identification of islet-cells (also those which occur outside the islets) takes on an eminently practical importance.
In the starfish Pisaster ochraceous, light microscopical histochemical analysis shows that the cells previously proposed as the source of starfish insulin are mucous cells secreting acid mucopolysaccharides into the pyloric lumen. No cellular or subcellular entity in the pyloric ceca satisfies the common staining criteria for β-cells, and the cytological source of starfish insulin is probably not demonstrable by conventional insulin stains. It is suggested that differentiated β-cells and granules may not occur in invertebrates and may be unique to the highly differentiated tissue systems of vcrtebrates.Evidence is presented that the paraldehyde fuchsin and pseudoisocyanin tests currently in common use for insulin detection are insufficiently trustworthy for use in diagnostic research on invertebrate or other inadequately analyzed tissues.
Administration of alloxan, which injures the ß cells and depresses insulin secretion, to animals with experimental hypothalamic obesity prevented any further increase in body weight despite an excessive food intake by the animals. It is concluded that the most important mechanism of development of obesity following injury to the ventromedial hypothalamic nuclei is the hypersecretion of insulin caused by such injury.
A light microscopic study of the endocrine pancreas in some myxinoids has revealed a striking difference in the occurrence of follicles. Islet tissue follicles are regularly occurring and also abundant in Myxine glutinosa, but are sparse or absent in Eptatretus burgeri and Eptatretus stouti. Whether this is caused by a decreased need for insulin in Myxine glutinosa is discussed but remains an open question.
Die B-Zellen des Inselapparates knnen mit einer Perameisensure-Eisenbindung/Sulfid-Silber Methode elektiv dargestellt werden. A-Zellen und exokrines Pankreas bleiben ungefrbt. Die Reaktion verluft, mehrstufig und beruht wahrscheinlich auf folgendem Prinzip: durch Perameisensureoxidation werden Disulfidgruppen des (Pro)Insulins oder dessen cystinhaltiger Vorstufen in stark saure Cysteinsulfonsuregruppen berfhrt, die kolloidales Eisen zu binden vermgen. Das gebundene Eisen wird mit Ammoniumsulfid und physikalischer Entwicklung in Form schwarzer Niederschlge dargestellt.B-cells of the pancreas are stained selectively with the performic acid-colloidal iron/sulfide-silver technique. A-cells and exocrine pancreas remain unstained. The rationale of the reaction depends on the oxidation of disulphide groups of (pro)insulin, or its precursors rich in cystine, to cysteine sulphonic acid groups. The free anionic groups so formed bind colloidal iron which in turn can be demonstrated by treatment with ammonium sulphide followed by physical development.
METHOD Experiments were carried out on 73 rabbits and 32 rats. Diabetes was produced in the rabbits by intravenous injection of 20-40 mg/kg dithizone in 0.25% ammonia solution. Tolbutamide was given by mouth in a dose of 500 mg/kg body weight to healthy animals and also to diabetic animals in different stages of the development of the disease. The blood sugar was determined by the Hagedorn-Jensen method before and 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 h after administration of the tolbutamide. The animals were sacrificed at the same times, the pancreas was fixed in Bouin's fluid and by Timm's method, and frozen sections of the pancreas were cut. The frozen sections, 10-20 p in thickness, were cut in a cryostat, and paraffin sections 5-10 # in thickness also were prepared. A histochemical reaction with zinc was obtained by means of 8-(p-tosylamino) -quinoline (8-TQ) and dithizone [3-5]. Acid phosphatase activity was determined at pH 5.0 by the azo-coupling method [7], carbonic and hydrase activity by a modified Kurata's method [14], glucose-6-phosph atase activity by Chiquoine's method [10, 11], and adenosinetriphos phatase activity by the method of Padykula and Herman [18, 19]. To detect acid phosphatase, frozen sections were fixed in 0.01% acetone solution of 8-TQ at 4~ for 30 rain, so that the reactions for zinc and the enzyme could be observed in the same section. The activity of the
Morphologic changes of the C-cells of 148 Wistar-rats were investigated by histochemical, cytophotometric and autoradiographic methods, and by electron microscopy. We studied normal animals aged 8 weeks to 2 years, acute or chronic hyper/hypocalcemic animals and animals fed with high/low calcium diet.
Spectrophotometric measurements in vitro with purified porcine calcitonin favour the assumption that PFA-PIC-staining makes possible the quantitative demonstration of calcitonin in the C-cells. On comparing the different experimental groups of animals a reduction of calcitonin content in C-cells was found in hypercalcemic animals. Increasing degranulation, seen by electron microscopy, corresponded with this finding. The calcitonin content was augmented in chronic hypocalcemic animals (because of parathyroidectomy). Bigger granules and lysosomes (Autophagolysosomes) were found electron optically. The number of C-cells increased in aged Wistar-rats until the appearance of C-cell-adenomas. The mitotic activity of C-cells found by3H-thymidin-autoradiography did not correspond with the level of serum calcium. The mitotic activity was intensified in animals fed with a low calcium diet; it diminished in chronic hypercalcemia. These findings are discussed concerning the regulation of calcium homeostasis.
Die Anfrbbarkeit der B-Zellen des Inselorgans mit der Eisenbindings-Reaktion wurde mittels der Standardmethode von Graumann bzw. Mowry untersucht. Es zeigte sich, da nach vorausgehender Schnittoxidation mit Perameisensure und einer Modifikation der Graumann'schen Methode eine selektive und sehr prgnante Darstellung der B-Zellen zu erzielen ist. Die Methode von Mowry erwies sich fr die Darstellung der B-Zellen als unbrauchbar. Der Nachweis der B-Zellen mit der Perameisensure-Eisenbindungs-Reaktion wurde mit Methoden bekannter Spezifitt (Aldehydfuchsin, Dichlorpseudoisocyanin) verglichen. Durch Umfrbeversuche konnte nachgewiesen werden, da mit allen drei Methoden jeweils identische Zelltypen (B-Zellen) dargestellt werden.The stainability of B-cells in islets of Langerhans by means of colloidal iron reaction has been examined using two standard modifications (Graumann resp. Mowry) of the original Hale-reaction. After previous oxidation of the sections with performic acid a strong and selective staining of B-cells was obtained by the use of a colloidal iron reaction based upon Graumann's method. With Mowry's technique B-cells remained unstained. The demonstration of B-cells using a performic acid-colloidal iron reaction has been compared with methods of known selectivity (Aldehyde Fuchsin, Dichlorpseudoisocyanine). By restaining procedures it could be shown that with the three methods the same type of cell i.e. B-cell is stained.
Die eingehende Untersuchung einer Reihe verschiedener Methoden hinsichtlich ihrer Eignung zum Nachweis von Granula in pankreatischen B-Zellen fetaler, jugendlicher und erwachsener Muse ergibt:1.
Die empfindlichsten Reaktionen sind bei Voroxydation diejenigen mit den Pseudoisocyaninen, ohne Oxydation die mit Aldehydfuchsin II und Crotonaldehydfuchsin. Weniger geeignet sind die Thiazine und Alcianblau, am wenigsten empfindlich erweist sich der SH- und SS-Gruppen-Nachweis nach Barnett und Seligman (1954).
2.
Die mit den Pseudoisocyaninen metachromatisch reagierenden - Granula sind optisch anisotrop.
A comparative study of various methods for detecting secretory granules of pancreatic beta cells in fetal, young, and adult white mice was carried out in order to find out the most sensitive reaction. The influence of prior oxidation with performic acid or permanganate was tested. The following results were achieved:1)
With prior oxidation (permanganate) the pseudoisocyanins, without oxidation aldehyde fuchsin II and crotonaldehyde fuchsin exhibited the highest sensitivity for beta granules. The thiazine dyes and alcian blue showed a lower sensitivity, whereas the method for demonstration of SH- and SS-groups according to Barnett and Seligman (1954) was proved to be less sensitive.
2)
Beta granules stained metachromatic with pseudoisocyanins were found to be anisotropic.
The general histological order, as well as size and localisation of Langerhans islets of the grass-snake Natrix n. natrix were investigated in the light and electron microscopes. In the light microscope, three main types of islet elements (B-, A1-, and A2-cells) and D- and amphiphil cells were identified. In the electron microscope B-, II-, III-, IV-, amphiphil-, and X-cells were identified, especially by the type of their secretory granules. The observations were related to results of previous studies on snakes and other vertebrates. The present study suggests that A1- and A2-cells may be identical with II- and III-cells respectively.Die Langerhansschen Inseln der Ringelnatter Natrix n. natrix wurden licht- und elektronenmikroskopisch untersucht. Beobachtungen ber die Lokalisation, die Gre und das histologische Gesamtbild der Inseln werden mitgeteilt. Lichtmikroskopisch wurden drei Haupttypen von Inselelementen gefunden, die bei allen Wirbeltierklassen vorkommen: B-, A1- und A2-Zellen, auerdem D-Zellen und amphiphile Elemente. Elektronenmikroskopisch wurden insbesondere nach dem Aussehen und der Gre der Sekretgranula Zellen der Typen B, II, III und IV identifiziert, auerdem noch X-Zellen und amphiphile Zellen. Es wird die Meinung vertreten, da die A1-Zellen mit Zelltyp II und die A2-Zellen mit Zelltyp III identisch sind. Die Ergebnisse werden mit Befunden, die bei Schlangen oder bei anderen Wirbeltieren erhoben wurden, verglichen.
A correlated morphological study, employing endocrine cell stains, immunofluorescence, and electron microscopy, was performed on biopsy specimens taken from a pancreatic tumor and liver metastases in a woman with hypoglycemic symptoms and high fasting insulin levels. The study revealed the tumor to be composed of two different endocrine cell populations, irrespective of the primary or metastatic growth. The first cell type fulfilled all the morphological characteristics of the islet B cells. The second was argyrophil (with the Grimelius silver method) and showed the morphological pattern of polypeptide-hormone-producing cells. With the lack of a detectable symptomatology, normal blood levels of the hormones other than insulin, and the negative results of a large number of immunofluorescence tests, we were unable to indetify the specific nature of the second type of cells.
The stainability of B-cells in islets of Langerhans by means of colloidal iron reaction has been examined using two standard modifications (Graumann resp. Mowry) of the original Hale-reaction. After previous oxidation of the sections with performic acid a strong and selective staining of B-cells was obtained by the use of a colloidal iron reaction based upon Graumann's method. With Mowry's technique B-cells remained unstained. The demonstration of B-cells using a performic acid-colloidal iron reaction has been compared with methods of known selectivity (Aldehyde Fuchsin, Dichlorpseudoisocyanine). By restaining procedures it could be shown that with the three methods the same type of cell i.e. B-cell is stained.
Behavior, pancreatic islets morphology and plasma glucose levels of male mice exposed for 2, 4, 24 and 48 hours to crowding stress were investigated. The crowding induced an intense turmoil state associated with enhanced irritability and aggressiveness among the specimens of all experimental groups. Violent fights occurred especially in the first 4--6 hours, generally with the death of 1--2 individuals from each group. The changes recorded in the pancreatic islets affected first (2 hours) exclusively the insulin-producing cells, and in subsequent intervals they progressively expanded over all cell types. The changes occurred during the experiment in all islet cell types; however the B-cells showed by far the most pronounced alterations irrespective of the studied time interval. Most changes suggested the stimulation of the entire gland secretory activity, but particularly of B-cells, which was also proved by low glycemia values recorded at 3 of the 4 crowding time intervals. On the other hand, some alterations, occurring first at 24 hours, were regarded as signs of a moderate B-cells secretory hypoactivity; they may partly support the slight hyperglycemia obtained at this time interval. The significance of the above short-term observations in the induction of glycoregulation disturbances, diabetes included, as well as the presumably mediation role of adrenal-cortex and -medulla hormones under stress conditions are discussed and correlated with findings reported in literature.
1.
The concentrations blood sugar and immunoreactive insulin decline during the first 5 h after VMHN lesion in rats. The reason for this is the very strong emotional and motor excitation.
2.
A subsequent two-hour consumption of food by animals with hypothalamic hyperphagia results in a more substantial increase in the blood sugar and insulin concentrations compared with control animals subjected to a sham operation.
Recent investigations strongly suggest the elaboration of a third pancreatic hormone by the D cell and the existence of cells which show the staining properties of both B and D cells. Demonstration of these and all other islet cells in a single section is possible by the following staining sequence: (1) of D cells by silver or toluidine blue, (2) of B cells by pseudoisocyanin, and (3) empirical staining of all islet cells together by aldehyde fuchsin, ponceau de xylidine, acid fuchsin and light green. Difficulties in embedding compact pancreatic tissue can be overcome by dehydrating to 80% ethanol, followed by tetrahydrofurane as the intermediate fluid to paraffin infiltration.
Aldehyde fuchsin, pseudoisocyanin and toluidine blue, histochemical dyes reported to be specific for insulin–containing granules of the pancreatic beta cell, were applied to insulin fixed in polyacrylamide gel by disc electrophoresis. Two major and four minor bands were resolved as demonstrated by staining with amidoschwarz; only the two major bands, were stained by aldehyde fuchsin. The addition of serum did not affect this reaction. Serum or insulin components gave no metachromatic reactions to the other stains. Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.
Over a period of six months four scimitar‐horned oryxes (Oryx tao) and one nilgai (Boselaphus tragocamelus) fell ill in the Amsterdam zoo. All showed glycosuria and concomitant polydipsia and polyuria. One other scimitar‐horned oryx manifested the same symptoms five years earlier.
Pancreatic atrophy in variable degree was found in all cases. Insulin in the β‐cells was demonstrated in minimal amounts in only one case. A comparison of the clinical symptoms and post‐mortem findings with published reports, possibly identical, in other animals did not lead to any final conclusion as to the etiology of the diabetes mellitus and pancreatic atrophy in the antelopes. It is possible that pancreatitis, overfeeding and genetic factors played a part.
Zusammenfassung
Diabetes mellitus mit Pankreasatrophie bei Antilopen im Zoo von Amsterdam
Innerhalb von 6 Monaten wurden 4 Oryxantilopen (Oryx tao) und eine Nilgai‐Antilope (Boselaphus tragocamelus) des Amsterdamer Zoos krank. Alle Patienten wiesen eine Glykosurie, Polydipsie und Polyurie auf. Eine Oryxantilope zeigte 5 Jahre früher die gleichen Symptome. In alien Fällen wurde eine Pankreasatrophie verschiedenen Grades gefunden. Nur in einem einzigen Fall konnten in den β‐Zellen minimale Insulinmengen festgestellt werden. Ein Vergleich der klinischen Symptome und der Sektionsbefunde mit früheren analogen Krankheitsberichten führte zu keiner definitiven Schlußfolgerung hinsichtlich Ätiologie des Diabetes mellitus und der Pankreasatrophie bei Antilopen. Pankreatitiden, Überfütterung und genetische Faktoren dürften vielleicht eine Rolle spielen.
Résumé
Diabète sucré avec atrophie du pancréas chez des antilopes du zoo d'Amsterdam
En six mois, quatre antilopes oryxes (Oryx tao) et un nilgaut (Boselaphus tragocamelus) sont tombées malades dans le zoo d'Amsterdam. Tous les animaux présentaient une glycosurie, ainsi qu'une polydipsie et une polyurie concomitante. Cinq ans plus tôt, une autre antilope oryxe avait présenté des symptômes analogues.
Dans tous les cas, on a observé une atrophie du pancréas à des degrés variables. Dans un cas, on a pu mettre en évidence des quantitéas trés faibles d'insuline dans les cellules β. Une comparaison des symptômes cliniques et des résultats de la nécropsie avec des rapports publiés sur des cas analogues ne permet aucune conclusion définitive sur l'etiologie du diabète sucré et de l'atrophie du pancréas chez les antilopes. Il est possible que pancréatite, suralimentation et facteurs génétiques aient joué un rôle.
Resumen
Diabetes sacarina con atrofia pancreática en antílopes del zoo de Amsterdam
En un período de seis meses cayeron enfermos cuatro antílopes oryx (Oryx tao) y un antílope nilgai (Boselaphus tragocamelus) en el parque zoológico de Amsterdam. Todos los pacientes presentaban glucosuria y polidipsia y poliuria concomitantes. Otro antílope oryx manifestó los mismos síntomas cinco años antes.
En todos los casos se halló atrofia pancreática en grados variables. En las células β se demostró insulina en cantidades mínimas en un solo caso. La comparación de los síntomas clínicos y de los hallazgos necrópsicos con informes nosológicos anteriores no condujo a ninguna conclusión definitiva con respecto a la etiología de la diabetes sacarina y la atrofia pancreática en los antílopes. Es posible que jueguen cierto papel las pancreatitis, super‐alimentación y factores genéticos.
Summary Four endocrine tumours of the pancreas associated respectively with the Zollinger-Ellison syndrome (1 case) and with hypoglycaemia
(3 cases) have been investigated by means of immunofluorescence, employing anti-insulin, anti-glucagon and anti-gastrin sera,
endocrine cell stains and electron microscopy.
In three cases the tumours were shown to be composed of different endocrine polypeptide cell types. The report examines the
role of the different investigative procedures in the recognition of endocrine tumours of the pancreas with multiple endocrine
polypeptide cell components.
Three main patterns have been recognized according to the distribution of the various endocrine cell types. The present findings
suggest in addition that different hormones are synthetized in different cells.
Chick embryos were exposed to a teratogenic dose of insulin at the 6-day incubation stage and the effect of this treatment on the histogenesis of the islets of Langerhans was studied throughout the incubation period. The exogenous insulin caused an initial delay in the differentiation of the beta islets and in the elaboration of the endogenous insulin. During the final prehatching stages, the endogenous insulin appeared to be elaborated more rapidly than in the control tissues. The insulin treatment caused an increased activity of the alpha cells and an increased production of glucagon. Histochemically, the ribonucleic acid content of both alpha and beta cells was reduced, especially during the early stages of development. Also, there was an initial increase in glycogen storage and alkaline phosphatase activity within the alpha cells. The beta islets in the insulin-treated tissues, except for the initial delay in their development, showed no significant differences in the glycogen and alkaline phosphatase patterns from the control.
1.
The cells of the peri-insular acini have larger nuclei and zymogen regions and are refractory to the secretion stimulating action of pilocarpine, in contrast to the cells of the common acini (the halo phenomenon).
2.
In addition to the data in the literature suggesting a relation between the presence of insulin producing B-cells in the islets and the halo phenomenon, we have shown that the destruction of the B-cells by alloxan results in a disappearance of the haloes.
3.
The peri-insular cells incorporate more leucine-3H than the cells of the common acini. The larger mass of ergastoplasm mainly accounts for this increased protein synthesis, the incorporation per unit volume of ergastoplasm not being substantially increased.
4.
This great protein synthesis in cells maximally filled with zymogen granules is in accordance with our finding (Kramer and Poort, 1968) that the rate of protein synthesis in the cells of the rat pancreas is a constant one, independent of the phase of the secretory cycle. As in common acini, 90 per cent of the protein synthesized during 45 min, in the peri-insular acini of a “resting”, non stimulated gland is lost from the cells within 24 hours. As much protein must leave the cell as is synthesized in order to prevent overfilling.
5.
A short discussion is given on the influence insulin can have on the secretion and on the protein synthesis in the acinar cells.
Repeated administration of xanthurenic acid to experimental animals completely eliminates insulin stores in pancreatic B cells
and attenuates normalization of blood glucose level in the glucose tolerance test. Xanthurenic acid inhibits insulin secretion
by isolated pancreatic islets and decreases zinc-specific luminescent reaction in B cells.
Mammalian pancreatic alpha granules were differentially stained with phosphotungstic acid haematoxylin. Paraffin sections were dewaxed and hydrated, oxidised 5-40 sec in freshly prepared 0.3% KMnO4 acidified with 0.3% (w/v) H2SO4, decolourised in 4% potassium metabisulphite, mordanted 20 min to 2 hr in 4% iron alum, stained in phosphotungstic acid haematoxylin 16-48 hr, rinsed in 95% ethanol until no stain runs from the tissue, dehydrated in absolute ethanol, cleared in xylene, and covered in synthetic resin. Advantages of this procedure are: (1) consistent, reproducible staining; (2) applicability to all the common laboratory mammals and man; (3) wide latitude at each stage, permitting its use as a routine method; and (4) superior visualization of alpha granules, due to suppression of background staining and absence of glare. For fixation, formalin-acetic or Bouin's solution is recommended.
1. Im Cytoplasma der insulinbildenden B-Zellen menschlicher und tierischer Pankreasinseln sowie in dem der sog. dunklen Zellen der Brockmannschen Krperchen der Schleie finden wir Granula, die mit Pseudoisocyaninfarbstoffen nach Oxydation metachromatisch reagieren.2. Reininsulin sowie alle bei der technischen Insulingewinnung aus Bauchspeicheldrsen entstehenden insulinhaltigen Fraktionen reagieren mit Pseudoisocyaninen nach Oxydation ebenfalls metachromatisch.3. Die metachromatische Reaktion des oxydierten Insulins bzw. der B-Zellstrukturen ist auf die Bildung von SO
3
–
-Gruppen zurckzufhren. Diese entstehen bei der Perameisensure- bzw. Kaliumpermanganatbehandlung durch oxydative Aufspaltung der Disulfidbrcken.4. Mit der Pseudoisocyaninreaktion, deren Durchfhrung genau beschrieben wird, wird das in den B-Zellen der Langerhansschen Inseln vorkommendeInsulin selbst nachgewiesen.5. Von den verschiedenen zur metachromatischen Darstellung der B-Zellen geeigneten Pseudoisocyaninen ist das N,N-Dithyl-6,6-dichlorpseudoisocyaninchlorid besonders zu empfehlen, da die mit diesem Farbstoff gefrbten Prparate mit Chloroform entwssert und in Caedax eingedeckt werden knnen. Auf diese Weise ist die Herstellung von Dauerprparaten mglich. Das leichter erhltliche N,N-Dithylpseudoisocyaninchlorid oder -bromid (Firma AGFA, Leverkusen) kann jedoch genauso verwendet werden, wenn die Prparate nach der Frbung und kurzem Wssern in wasserlsliche Eindeckmittel eingeschlossen werden.6. Die Pseudoisocyaninreaktion besitzt fr den Insulinnach weis einehohe Spezifitt. Nur Proteine, bei deren oxydativer Behandlung wenigstens zwei unmittelbar benachbarte SO
3
–
-Gruppen (Abstand 4–5 ) gebildet werden, reagieren metachromatisch. Eine derartige Gruppierung liegt bei der durch die Perameisensure-Oxydation von Insulin entstehenden Polypeptidfraktion A vor. Fraktion B, bei der die SO
3
–
-Gruppen rumlich weit entfernt sind, reagiert negativ. Ebenso ist mit unoxydiertem und oxydiertem Oxytocin, Glutathion und Eieralbumin sowie Aminosuren, unter ihnen Cystin, Cystein und Cysteinsure, ihr Oxydationsprodukt, keine metachromatische Reaktion zu erzielen.7. In den B-Zellen von oxydierten Schnitten durch Pankreasinseln fehlt bei der Frbung mit anderen als metachromatisch bekannten Farbstoffen (Toluidinblau, Methylenblau, Acridinorange) eine metachromatische Reaktion. Dies wird darauf zurckgefhrt, da mit Pseudoisocyanin im Gegensatz zu den anderen metachromatisch wirkenden Farbstoffen bereits beim Vorliegen von Substanzen mit nur zwei oder wenigen negativen Gruppen Metachromasie zu erzielen ist.8. Die Darstellung von Insulin im oxydierten histologischen Prparat mit Hilfe der Pseudoisocyaninreaktion gelingt nur nach Fixierung mit formalinhaltigen Gemischen, jedoch nicht nach Alkohol- oder Acetonfixierung. Mglicherweise wird Insulin aus alkohol- oder acetonfixiertem Gewebe durch die sauren Oxydationsmittel herausgelst, da diese Fixierungsmittel lediglich eine Eiweifllung bewirken. Bei Verwendung von formolhaltigen Gemischen kann dagegen durch Vernetzung von Insulin mit anderen Proteinen ein sureunlsliches Kondensationsprodukt entstehen.1. The cytoplasm of the insulin-producing B-cells of the islets of Langerhans contains granula which show a metachromatic reaction after oxidation and colouring with pseudoisocyanins. Such granula can be found in human and mammalian islets, as well as in the insulin-producing tissue of a fish, Tinca vulgaris.2. Pure insulin as well as all insulin containing fractions obtained during the process of technical isolation of insulin react metachromatically with pseudoisocyanines when oxidized prior to the colouring.3. The metachromatic reaction of oxidized insulin and of some cytoplasmic structures of B-cells is due to the presence of SO
3
–
-groups. They are formed by the splitting of disulphide bonds on treatment with performic acid or potassium permanganate.4. The metachromatic reaction with pseudoisocyanins, which is described in detail, can be used for thedetection of insulin in the islets of Langerhans.5. Several pseudoisocyanins can be employed for the histochemical colouring of B-cells. NN-Dithyl-6,6-dichlorpseudoisocyaninchlorid, however, is the most suitable compound since it allows the dehydration of the slides with chloroform after staining. Coverslips can than be mounted with Caedax and the preparates can be made permanent. NN-Dithylpseudoisocyaninchlorid, which can be obtained more easily since it is produced commercially by AGFA-Leverkusen, can also be used, but after staining and rinsing in water the slides must be mounted with a water soluble medium.6. The reaction with pseudoisocyanins ishighly specific for the detection of insulin. Only proteins, which are characterised by at least two closely neighbouring SO
3
–
-groups (distance about 4–5 ) react metachromatically. Such a configuration of SO
3
–
-groups occurs in the polypeptide fraction A, which is formed by oxydation of insulin with performic acid. Fraction B does not react metachromatically since its SO
3
–
-groups are not located closely enough to each other. No metachromatic reaction is seen after staining of either unoxydized or oxydized oxytocine, glutathione, eggalbumen and amino acids such as cystine, cysteine or its oxydized form, cysteinic acid.7. B-cells of oxydized slices of pancreatic islets show no metachromasia with other dye stuffs such as toluidine blue, methylen blue and acridine orange, which are commonly known for their metachromatic effects. It is believed that pseudoisocyanine reacts metachromatically already with two negative groups, whereas the other dyes require a greater number of negative groups.8. Insulin can be characterized in oxydized histological slides only after fixation with formaline-containing solutions, but not after fixation with alcohol or acetone. Since alcohol and aceton merely precipitate the proteins, it can be assumed, that the insulin is eluted by the acidic oxidants when alcohol and acetone have been used for fixation. Formalin on the other hand can react with insulin and other proteins and build up an acid-insoluble condensation product.
A comparison is made of the relative affinities of aldehyde-fuchsin and the Schiff reagent for various tissue elements in different states of oxidation.
Glycogen, gastric mucus, kidney brush border, intestinal striated border, reticular tissue, gastric parietal cell canaliculi, and Paneth cell granules react with neither aldehyde-fuchsin nor the Schiff reagent without periodic acid or stronger oxidation.
Beta cell granules of the islets of Langerhans, keratin, mast cells, elastic fibers, hyaline cartilage, goblet cells, thyroid colloid, argentaffine cell granules, and the acrosomes of spermatozoa are stained by aldehyde-fuchsin without oxidation. None of these tissue elements exhibit a clearly positive reaction with the Schiff reagent under similar conditions. However, elastic fibers, hyaline cartilage, and acrosomes do display a slight coloration.
Since the tissue elements which do not stain with aldehyde-fuchsin without previous oxidation act in a similar manner toward both aldehyde-fuchsin and the Schiff reagent, it is assumed that aldehyde groups are responsible for the positive reaction with both these reagents after periodic acid or stronger oxidation.
The tissue elements which are stained most intensely by aldehyde-fuchsin without oxidation, i.e., mast cells, hyaline cartilage, and elastic fibers, are those which have been found to contain or to be associated with highly sulfated mucopolysaccharides.
Beta cell granules and keratin which after oxidation react strongly with aldehyde-fuchsin, but not with the Schiff reagent, are known to possess a high content of cystine.
Since the sulfated mucopolysaccharides possess sulfuric groups and since the dithio bonds of cystine are converted to sulfonic groups on oxidation, it is suggested that these groups may be responsible for the staining of tissue elements by aldehyde-fuchsin when the Schiff reaction is negative.
In conclusion, it appears that the reactivities of aldehyde-fuchsin and the Schiff reagent are similar in that they both show an affinity for aldehyde groups, but different in that aldehyde-fuchsin seems to possess an affinity for strong sulfur acids which the Schiff reagent does not.
WITH the development of a new specific method for the histochemical demonstration of protein-bound sulfhydryl groups (Barrnett and Seligman, 1952), and disulfide groups (Barrnett and Seligman, 1954), the way was opened for the demonstration of individual proteins particularly rich in these reactive groups or of regions in tissue where such proteins were concentrated. The success or failure of such a demonstration would depend, in part, upon whether solution and extraction of the protein under the conditions of the method could be avoided.
In order to develop a method for demonstrating insulin in the islets of Langerhans, in various species and in various physiological states, we were encouraged by the facts that insulin, although deficient in sulfhydryl groups, is rich in disulfide by virtue of its 12% cystine content (du Vigneaud, Miller, and Rodden, 1939), that insulin was available in pure crystalline form for critical in vitro experiments to work out the details of the histochemical procedure
Since Moloney and Coval1 have demonstrated that antibodies to insulin can be produced in the guinea pig, it seemed feasible that these antibodies could be labeled with fluorescein isocyanate by the method of Coons and Kaplan2 and used as an immunochemical stain for insulin. The results obtained from the application of these labeled antibodies to frozen sections of pancreas are reported in this paper.
A method for the staining of mammalian pancreatic islets with a specific fluorescent antibody to insulin using frozen-dried sections rather than those cut with a cryostat is described. Sections were cut on a Servall microtome at 1-2 μ or when embedded in paraffin they were cut at 4-5 μ on a conventional rotary microtome. The latter procedure greatly simplified the search for islets.
This chapter focuses on the accuracy of the peptide theory and on the possible existence of non- peptide bonds in proteins. Partition chromatography in the form of paper chromatography is a method that is identified as breakdown products of proteins where more peptides are used. This technique had been identified by the classical methods of organic chemistry. Three types of polypeptide chains are possible, open, cyclic and branched. There are two types of terminal residues, those with a free amino group and those with a free carboxyl group. The Dinitrophenyl (DNP) method is used for the separation and identification of peptides containing lysine. Elution can be effected with acid ethanol and the peptides subsequently fractionated on suitable partition chromatograms. The theoretical possibility exists that synthetic reactions may occur during the partial hydrolysis of proteins. Acid hydrolysis is the most used method of degrading proteins, and it is almost universally employed when amino acids are to be isolated or estimated, since it leads to complete hydrolysis with a minimum of destruction. Paper chromatography is preferred for the final fractionation of a simplified peptide mixture because of the good resolutions obtained, the ease and rapidity of technique, and the possibility of using the two-dimensional method. The chief disadvantage is that only a very small amount of each peptide can be obtained from a chromatogram, though it is usually possible to obtain sufficient material to determine the structure of di- and tri-peptides.
Zinc-dithizone reaction of pancreatic islets Oxidation of insulin in performic acid Fractionation of oxidized insulin
- W F Mcnary
McNary, W. F. 1954. Zinc-dithizone reaction of pancreatic islets. J. Histochem. Cytochem., Sanger, F. 1947. Oxidation of insulin in performic acid. Nature, 160: 295-6. __ 1949. Fractionation of oxidized insulin. Biochem. J.. 44: 126-8.
The Metachromatic Reaction. Protoplasmatologia Bd
- J W Kelly
Kelly, J. W. 1956. The Metachromatic Reaction. Protoplasmatologia Bd. II/D 2. Springer Verlag, Vienna.
Über den Nachweis von Insulin mit den metachromatische reagierenden Pseudoisocyaninen
- T H Schiebler
- S Scheissler
- Sanger F
Extractable insulin of pancreas
- G A Wrenshall
- A Bogoch
- R C Ritchie