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Dichromatism of bromphenol blue, with an improvement in the mercuric bromphenol blue technic for protein.

Staining of protein in sections using the mercuric bromphenol blue technic is improved by staining with 1% HgCl2 and 0.05% bromphenol blue in 2% aqueous acetic acid for 15 min at room temperature. Rinse slides 20 min in 2 changes of 0.5% aqueous acetic acid. Blot and give 2 fast changes in absolute ethanol with agitation before transferring to xylene. Transfer slide to 0.5% n-butylamine in xylene for a few seconds until the section is blue, then, after 2 changes of xylene, mount in DPX. Spectrophotometric analysis of this blue dye at different concentrations and with or without heparin showed that the reddish hues are due to dichromatism and not metachromasia.

Animals

Enhancement of pulmonary drug absorption in the rat by bromphenol blue and related dyes.

1. Pulmonary absorption studies in the rat showed that intratracheally administered 5-10 mM bromphenol blue, bromcresol green and bromthymol blue markedly increased the absorption rate of 0.1 mM phenol red. 2. Similarly, 1-10 mM bromphenol blue increased the absorption rate of 0.1 mP p-,minohippuric acid, tetraethylammonium and mannitol by 2- to 18-fold in a concentration-dependnet manner. 3. Mannitol absorption was enhanced more by bromthymol blue, sulphobromophthalein, bromcresol purple, thymol blue and bromcresol green than by bromphenol blue or m-cresol purple. Chlorphenol red and phenol red had no effect on mannitol absorption. 4. The results indicated that certain sulphonic acid dyes increase the permeability of the respiratory tract epithelium, perhaps by increasing its porosity.

Aminohippuric Acids

[Determination of residual bilirubin binding capacity in the serum of newborn infants using bromphenol blue].

In serum specimens collected from premature infants the residual bilirubin binding capacity was determined by a direct spectrometric method using bromphenol blue. The bindingin capacity determined in this way is strongly correlated to the serum bilirubin level. Changes in binding capacity are shown during the first 16 days of life. Experiments on model sera show the dependence of binding capacity for bromphenol blue on the ratio albumin/bilirubin. The results are discussed with respect to the urgency of blood exchange transfusions preventing danger of kernicterus.

Bilirubin

Available bilirubin binding sites of serum from newborns determined by a direct spectrometric method using bromphenol blue.

A new direct spectrometric micromethod for the determination of the available bilirubin binding sites of serum (S-ABBS) using bromphenol blue has been applied to 298 blood specimens collected from infants immediately before starting the first exchange transfusion. A relative number fraction of 0.07 of the material, characterized by having the lowest S-ABBS values, includes seven of the nine cases (i.e. a relative number fraction of 0.78) with autoptically verified kernicterus (KI). The two infants with KI but higher S-ABBS values differed in having severe neurological symptoms before the examination. The mean substance concentration of bilirubin in these nine sera was 288 mumol/l. The correlation of KI to the body mass at birth, to the substance concentration of serum albumin, and to the substance concentration of serum bilirubin is given, and the sources of error concerning the method are discussed. The present method is proposed for determining a type of quantity that is important, among other types of data, in deciding on the need for exchange transfusion in icteric (premature) newborns.

Bilirubin

A direct spectrometric method for determination of the concentration of available bilirubin binding sites in serum using bromphenol blue.

A simple and rapid direct spectrometric micromethod for the determination of the concentration of available bilirubin binding sites of serum is given. The method measures - by means of difference spectrometry - the maximal change in absorbance when serum is added to a buffered solution of bromphenol blue (BPB). This dye fulfills a set of requirements given and examined. The binding properties of bilirubin, BPB, and some related dyes are spectrometrically investigated, and differences between the properties of human and bovine albumin and between such albumin preparations and human serum are shown and discussed.

Adult

[Essay of proteins after aldehyde treatment in biological objects (author's transl)].

Following fixation with formalin or glutaraldehyde, the protein content of bovine serum albumin, lysozyme, non-disintegrated yeast cells and liver nuclei of mice is determined by means of a brom-phenol blue staining procedure and the widely used Lowry-technique. The bromphenol blue technique permits determinations of soluble as well as particulate proteins or protein mixtures fixed with up to 6% formalin or 2% glutaraldehyde. Recovery rates differ no more than 20% as related to unfixed controls. Using the bromphenol blue method it is not necessary to separate aldehyde or any interfering material by additional steps prior to determination. Factors for correcting protein content of biological material after aldehyde treatment are available. In this way, comparative biochemical as well as cyto- and histochemical investigations of enzymatic activities after aldehyde fixation are possible. Some advantages of the bromphenol blue technique with special reference to the analysis of particulate and/or aldehyde-fixed specimens are discussed.

Animals

[Comparative evaluation of modern intraoperative diagnostic methods in malignant gliomas of the brain].

The following methods were employed: Intravital staining with bromphenol blue (70 patients), radiometry with P 32 (127 patients), echo encephalography (12 patients,) puncture biopsy (18 patients), rheometry (96 patients). Bromphenol blue is injected into the a. cerebri media or the supraclinoidal a. carotis interna and stains the periphery of malignant tumours and the entire tissue of benign tumours. Radiometry also serves for the intraoperative finding of tumour remainders which must be located at least 20 to 22 mm below the cortex of the brain. Tumour biopsy was carried out with a sharp cannula to avoid deformation of the tissue cylinder. Rheometry proved to be very useful when avoiding the mixing with blood, liquor, and cyst fluid at the measuring tip.

Biopsy, Needle

[Coelomic gonadal epithelia during the period of human embryonic morphologic sex differentiation].

The course of divergent differentiation of coelomic epithelium in the indifferent gonad has been traced up to the stage when morphological sex signs are distinctly seen. Eleven human embryos fixed in Carnoy and Bouin's fluid have been studied from 6 to 10 weeks of gestation. Paraffin sections were stained with hematoxylin, ferric hematoxylin after Heidenhain. Basal membrane fibres were revealed by silver impregnation after Karupu. PAS-reaction after McManus and additional staining with hematoxylin and light green were applied, as well as the reaction for total proteins with bromphenol blue after Miguel--Calvo. Sex differentiation of coelomic epithelium in the gonad developing according to the male type begins a little later than in the ovary. In the epithelium of the forming testis a great amount of mitotic figures is observed that might be connected with the presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing number of mitotic figures in the epithelium should be considered as a sign demonstrating that differentiation according to the male type has started. Superficial epithelium of the embryonic ovary has a peculiar false pseudostratified structure. This secures cellular reserve for the ovarian cortex formation from the external epithelium. The apical surface of the cells in the external epithelium has a border which is evidently formed by microvilli and revealed by PAS-reaction and bromphenol blue. Ovarian follicular cells and Sertoli's cells (sustentocytes) in testis have the common origin in the human fetus--from coelomic epithelium of the gonad germ.

Epithelial Cells

Procedure for calibrating the Technicon Colorimeter I.

We describe a rapid method for calibrating the Technicon AutoAnalyzer colorimeter I. Test solutions of bromphenol blue are recommended for the calibration, in preference to solutions of potassium dichromate, based on considerations of the instrument's working range and of the stray light characteristics of the associated filters.

Autoanalysis

[A simple qualitative method for assessing microalbuminuria in diabetics].

Diabetes mellitus is one of the most important causes of kidney diseases, the first sign of which is albuminuria. It is now possible to detect microalbuminuria before the appearance of larger quantities. We compared a simple qualitative test for microalbuminuria (change in color of a tablet containing bromphenol blue on addition of urine) with a more elaborate quantitative immunoturbidometric method. Of 109 samples tested 31 were positive by the quantitative method and 29 of these were positive by the tablet method. Out of the 78 negative by the quantitative method only 4 were positive by the qualitative test. The sensitivity of the tablet test was 93.5%, specificity 94.0%, positive predictive value 87.9%, negative predictive value 97.4%, false positive rate 5.2%, and false negative rate 6.2%. We conclude that the tablet test is simple and accurate and is suitable both for screening of large populations and for the diagnosis of individual cases of microalbuminuria.

Albuminuria

An anomalous effect of the intermediate products of riboflavine photolysis on the intestinal absorption of poorly absorbed water-solbule drug in rats.

The influence of the intermediate products of riboflavine photolysis on the absorption of poorly absorbed water-soluble drugs from the rat small intestine has been examined using an in situ recirculation technique. The absorption of phenol red, bromphenol blue (BPB) and their biliary excretion, and lactose isonicotinoyl-hydrazone (lactose-INH) and its plasma concentration were increased in experiments made in the presence of light in contrast to other made in the dark. The absorption of both phenol red and lactose-INH were concentration-dependent. On the other hand, a saturation phenomenon was demonstrated when the concentration of phenol red was kept constant while that of the water-soluble compound flavine mononucleotide (FMN) was changed. The results which were obtained from pretreatment experiments suggest an alteration in the permeability of the intestinal membrane. No enhancement effect could be demonstrated for lumichrome in the presence of absence of light.

Animals

Appearance of free histones within nuclei of differentiating cells in the developing rat embryo.

Having applied the picric acid-bromphenol blue (BPB) and the phosphomolybdic acid-benzidine (PMA-B) methods--the first method detecting the total histone content, the second one only free histones--we have found that cell nuclei of the rat embryos permanently contain histones, nevertheless, in such a pattern that the free histone appears only at a well-defined stage of embryogenesis. Strong PMA-B reaction is observable in the nuclei of some mesenchymal cells and in the reorganizing somites' cell nuclei of the 13-day-old embryo as well as in the hemopoietic elements of the 15- and 17-day old embryos' liver. Within nuclei of the red blood cells of the 13-day-old embryo an extremely intense reaction is seen which then disappears till the 15th day, while the BPB reaction shows a gradual intensification up to the neonatal age. The experiments support the former model of one of the authors for a histone-nonhistone control of ontogenesis.

Age Factors

Histochemical demonstration of hyaluronic acid in human dermal mast cells.

Acid mucopolysaccharides in mast cell granules were histochemically studied in the lesion of urticaria pigmentosa and in the dermis of normal human skin. Alcian blue and azure A were used to stain mucopolysaccharides. Bromphenol blue was employed for detection of basic proteins. In a further attempt to identify various polyanions, staining was carried out with alcian blue containing various concentrations of electrolytes. Methylation, saponification, and digestion with streptomyces or testicular hyaluronidase, chondroitinase ABC, sialidase, or desoxyribonuclease were also employed. The results obtained are most likely to suggest the presence of hyaluronic acid in mast cell granules.

Histocytochemistry