Unconjugated bilirubin in human bile: the nucleating factor in cholesterol cholelithiasis?

AIMS: To investigate the concentrations of bilirubin, bilirubin conjugates, phospholipid, and cholesterol in the gall bladder bile obtained at surgery from patients with and without cholesterol gallstones. METHODS: Gall bladder bile was collected during surgery, by puncture, from 20 patients with gallstones undergoing routine cholecystectomy and from eight patients with normal liver blood tests. Concentrations of bilirubin, bilirubin conjugates, phospholipid, and cholesterol were measured using standard procedures. RESULTS: The proportion of total bilirubin that was unconjugated was significantly higher in the bile from patients with stones than in bile from control patients, whether or not the bile from either group was saturated with cholesterol or not. Indeed, the mean concentration of cholesterol was significantly higher in control bile samples. CONCLUSION: The presence of stones was more closely related to the proportion of unconjugated bilirubin than to the degree of saturation of bile with cholesterol. Bilirubin and its metabolites probably play an important part in the formation of cholesterol gallstones.

Sampling variability of liver biopsy in inflammatory bowel disease.

Sampling errors may occur when hepatic needle biopsy specimens are obtained for histologic examination. We obtained biopsy specimens from the right and left lobes of the livers of 29 patients who were to undergo surgery for inflammatory bowel disease. The histologic appearances of Kupffer's cell hyperplasia, fatty change, portal tract inflammation, and focal necrosis, but not giant cell granulomas, were well represented by a single specimen. The incidence of minor hepatic abnormalities was high and is likely to reflect the severity of the disease leading to surgery, but serious lesions were uncommon.

Specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine.

This paper presents methods for specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine in tissue sections from which RNA has been extracted selectively with concentrated phosphoric acid at 5 degrees C for 20 min or by hydrolysis in 6 N HCl at 28 degrees C for 15 min. It has been found that pH of the freshly prepared celestin blue B dye solution is 3.0 and that of an aqueous solution of gallocyanine is 2.8. These pHs can be lowered to 1.5 with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But if the pHs are lowered with concentrated hydrochloric or phosphoric acid, effective use of these dyes is not possible. It has been suggested that some dispersion of the two dyes takes place with concentrated sulphuric or nitric acid which are used to lower the pH. Staining of the nuclei is also possible with an aqueous solution of celestin blue B at pH 3.0 but the same is not possible with gallocyanine at pH 2.8. The absorption spectra of nuclei stained with an aqueous solution of celestin blue B at pH 1.5 and 3.0 are fairly identical, the peak of maximum absorption being at 620 nm. Those of nuclei stained with an aqueous solution of gallocyanine reveal irregular peaks. Possible implications of these findings have been discussed.

Staining of acid-hydrolysed tissue sections with Schiff-type dye-reagents under UV rays.

This paper reports on the use of some Schiff-type dye-reagents prepared with oxalic acid and sodium thiosulphate. Some of the dyes, such as azure A, azure B, toluidine blue O, thionine, brilliant cresyl blue, and methylene violet were also prepared with oxalic acid and sodium thiosulphate but fortified with disodium hydrogen phosphate. It has been found that all these dye-reagents, barring neutral red-SO2, when used on acid-hydrolysed mammalian tissue sections under UV rays yield far better staining of the DNA-aldehyde molecules than is possible in controls stained under normal laboratory conditions. Sections stained with any of the dye-reagents under UV rays do withstand treatment in SO2 water or acid water without showing any leaching of the dye from the nuclei. Aqueous solutions of all these dyes can also be used to stain DNA-aldehyde molecules of acid-hydrolysed tissue sections. But sections stained with aqueous solutions of the azures, safranine, phenosafranine, and methylene violet when treated with SO2 water reveal considerable leaching of the dye from the nuclei. However, leaching does not occur when these dyes are used as SO2-containing dye-reagents under UV rays. It has, therefore, been concluded that in the case of staining with the azures, safranine, phenosafranine, and methylene violet, containing SO2, the reaction under UV rays is of Feulgen-type. In the case of staining with toluidine blue-SO2, thionine-SO2, and brilliant cresyl blue-SO2 under UV rays, the mechanism of staining is also of Feulgen-type but it is due to liberation of the dye molecules from their more or less colourless states that react with the DNA-aldehyde molecules producing nuclear colouration as though these dyes are in aqueous solution. This interpretation is based on the fact that sections from which RNA has been extracted selectively with cold concentrated phosphoric acid when treated with these dye-reagents under UV rays also stain DNA-phosphate groups. The increased staining intensity of the nuclei produced with the majority of the dye-reagents under UV rays as compared with control sections stained under usual laboratory conditions has been considered to be due to electronic excitation of the dye molecules thus facilitating binding between more molecules of DNA-aldehyde and of dye. Possible significance of these findings has been discussed.

Basic dyes for the staining of DNA in mammalian tissues and absorption spectra of stained nuclei in the visible light.

This communication presents informations on the staining of DNA with basic dyes, such as ethyl violet, Janus green, a new red dye obtained from Janus green, and Giemsa, belonging to different chemical groups. It has been found that DNA of tissue sections from which RNA has been extracted selectively with cold concentrated phosphoric acid can be stained with aqueous solutions of these dyes. Further, DNA of tissue sections from which RNA has been extracted and the sections then hydrolysed in 6 N HCl at 28 degrees C for 15 min can also be stained with aqueous solutions of ethyl violet, Janus green, and Giemsa. Moreover, tissue sections that have been hydrolysed in 6 N HCl and then stained with aqueous solutions of these dyes, including the new red dye obtained from Janus green, reveal well-stained nuclei. The absorption spectra of nuclei stained with aqueous solutions of ethyl violet, Janus green and Giemsa, following the above stated three procedures of staining have been presented. Absorption data of nuclei stained with the new red dye after Feulgen hydrolysis of tissue sections as well as those of nuclei in tissue sections from which RNA has been extracted have also been presented herein. The implications of these findings have been discussed.

Saline flush: a simple method of reducing diazepam-induced thrombophlebitis.

Five hundred outpatients undergoing endoscopy were admitted into a controlled trial comparing the incidence of thrombophlebitis following intravenous diazepam administered in the way, with the effects of either a saline flush following the diazepam or diluting the drug with the patient's own blood before injection ('barbotage'). The results were assessed using a questionnaire completed by patients two weeks after endoscopy; 80% replied. A saline flush reduced the incidence of side effects, particularly pain (P less than 0.05). 'Barbotage' gave the highest incidence of side effects. Saline flush is therefore recommended as a means of reducing the thrombophlebitis which may follow intravenous diazepam.

Use of aqueous solutions of two basic dyes for the demonstration of DNA.

This paper presents informations as to the ability of aqueous solutions of two basic dyes, such as Dahlia and Victoria blue, belonging to aminotriarylmethane group for the staining of DNA-aldehyde molecules as well as DNA-phosphate groups. It has been found that sections of rat tissues stained with aqueous solutions of these dyes after acid hydrolysis followed by drying between folds of filter paper and treatment in n-butanol for a minute and then by a very brief treatment in a mixture consisting of equal parts of n-butanol and absolute ethanol reveal well-stained nuclei. Tissue sections after acid hydrolysis when stained with aqueous solutions of these dyes and then treated with SO2 water do not reveal any colouration of the nuclei. Since both the dyes are without any primary amino group in their molecules, it has been concluded that the imino group of Dahlia and the tertiary amino group of Victoria blue with cold concentrated phosphoric acid and then stained with any of these dyes also exhibit well-stained nuclei. The absorption spectra of nuclei stained with these dyes for DNA-aldehyde molecules as well as DNA-phosphate groups reveal positions of the peaks of maximum absorption at the same wavelength, which, however, are different in the case of nuclei stained with the two dyes. The implications of these findings have been discussed.

Basic dyes in the staining of DNA-phosphate groups and DNA-aldehyde molecules in cell nuclei.

A novel method is presented for the staining of cell nuclei with aqueous solutions of Janus blue, methylene blue, and Janus red in tissue sections from which RNA has been extracted selectively with cold phosphoric acid. Not only this, DNA-aldehyde molecules can also be stained when tissue sections from which RNA has been extracted are then hydrolysed in 6 N hydrochloric acid at 30 degrees C for 15 min followed by staining with Janus blue, methylene blue, and Janus red. Following staining with any of these dyes, sections can be dried between folds of filter paper and then treated with n-butanol or passed through grades of ethanol, cleared in xylene and mounted. Staining with Janus blue has been considered to be metachromatic, particularly in the sections of the rectum in which glycogen stains blue-black and the nuclei purplish. An aqueous solution of methylene blue does also stain glycogen blue with similar colour of the nuclei while with Janus red both the nuclei as well as glycogen stain red. The in situ absorption spectra of the nuclei stained with the dyes mentioned, after selective extraction of RNA, reveal peaks of maximum absorption at 560-570 nm (Janus blue), at 600 and 640 nm (methylene blue), and at 530 nm (Janus red). Those of nuclei stained for DNA-aldehyde molecules are at 560 nm (Janus blue), at 600 and 630 nm (methylene blue), and at 520 nm (Janus red). Possible significance of these findings has been discussed.

Computed tomography and EEG in herpes simplex encephalitis. Their value in diagnosis and prognosis.

Herpes simplex encephalitis (HSE) is associated with high mortality and morbidity, but these rates may be reduced by early administration of vidarabine. In six of 11 patients with the disease, the computed tomographic (CT) scan was of diagnostic value and showed low-density areas primarily in the temporal lobes; in only two of these six patients did the EEG point to the true diagnosis. Two of the six died. In the other five patients, the CT scan was normal or showed minor changes but the EEG was grossly abnormal. All five of these patients died. Information from CT scanning and the EEG should be combined for early diagnosis in HSE.

Gallamine blue staining of DNA in mammalian tissue sections: analyses of in situ absorption spectra.

This paper reports on the use of gallamine blue (GB), a dye of the oxazine group, as a specific stain for DNA in animal tissue nuclei. The dye can be used as 1% aqueous solution in boiling distilled water at ph 1.0 to 1.5. Not only this, GB dye-reagent can also be prepared after dispersing the dye with concentrated sulphuric acid and then dissolving the friable mass in 1% cobalt chloride and then used to stain nuclei at very low pH. Although the dye does not contain any primary amino group in its molecules, it can be used as aqueous solution or as dye-reagent to stain DNA-aldehyde molecules in tissue sections which are hydrolysed in 6N HCl at 28 degrees C or at 40 degrees C for 15 and 5 min, respectively. Following staining of the DNA-aldehyde molecules, the preparations cannot be treated with SO2 water, since this treatment brings about complete leaching of the dye from the nuclei. It has, therefore, been concluded that GB staining of DNA-aldehyde molecules is due to a modified Feulgen reaction in which tertiary amino group may be involved. Moreover, GB in an aqueous solution or as a dye-reagent can be used to stain DNA-phosphate groups in tissue sections from which RNA has been extracted selectively with cold concentrated phosphoric acid. Sections from which RNA has been extracted and then hydrolysed in 6N HCl at 28 degrees C or at 40 degrees C for 15 and 5 min, respectively, can also be stained with this dye. The absorption spectra of nuclei stained following the various procedures have been presented. The paper contains a discussion on the implications of all these findings.

Phosphoric acid-its use in the extraction of RNA: staining of DNA in mammalian tissue sections.

This communication presents highly satisfactory methods for the demonstration of DNA in animal materials. The method involves selective extraction of RNA with concentrated, 90% or 75% phosphoric acid of 5 degrees C for 20, 40 and 120 min, respectively, followed by staining with aqueous solutions of basic dyes, such as setoglaucine, setocyanine, pinakryptol green 2) and alcoholic aniline blue without SO2. Perfect blue nuclei were seen when staining was performed with aqueous solutions of setoglaucine and setocyanine at pHs 3.5 and 4.0 to 4.5, respectively. Sections of tissues from which RNA has been extracted and then hydrolysed in 6 N HCl at 28 degrees C for 15 min followed by staining with these dyes also revealed perfect colouration of the nuclei. Acid-hydrolysed sections when stained with alcoholic aniline blue-SW2 prepared with N HCl and sodium thiosulphate revealed nuclei of magenta colour, and sections from whcih RNA has been extracted and then hydrolysed in hydrochloric acid and stained with this dye-reagent revealed nuclei of purplish colour. Sections of tissues fixed in Carnoy, 10% buffered neutral formalin as well as paraformaldehyde were found to be most suitable for staining with these dyes. The in situ absorption spectra of nuclei stained with aqueous solutions of setoglaucine, setocyanine and alcoholic aniline blue without SO2, after extraction of RNA as well as those of nuclei in tissue sections from which RNA has been extracted and then acid-hydrolysed and stained with alcoholic aniline blue-SO2 have been presented. Also presented herein are absorption data of nuclei in tissue sections which wee hydrolysed in hydrochloric acid and then stained with alcoholic aniline blue-SO2. Some implications of these findings have been discussed.

Meta-phosphoric acid-its use in the selective extraction of RNA from fixed tissue sections.

This communication presents a new quick method for selective extraction of RNA from formalin-fixed tissues, such as kidney, intestine, ovary and testis of white rat and liver of rodents, Tatera indica and Millardia meltada and of the frog, Rana tigrina as well as of human wart. Sections of pancreas and kidney fixed in acetic acid-alcohol were also tried. The method is to treat deparaffinised sections in 20% and 15% meta-phosphoric acid at 5 degrees C for 10-20 and 20-30 min, respectively, and then to stain nuclear DNA with 0.5% aqueous solution of methyl green, methylene green, Giemsa, toluidine blue O or 0.125% pyronin G for 2 min, rinsed with water, treated with n-butanol for 2-3 min, cleared in xylene and mounted. It has been found that liver sections of rodents require 24 hours of treatment in 15% cold meta-phosphoric acid for complete removal of RNA, whereas those of the frog require only 20 min at 20% acid. It has been concluded that following treatment of sections in cold meta-phosphoric acid, RNA is extracted selectively leaving DNA in a native state. Therefore, staining of DNA in tissue sections from which RNA has been extracted is due to binding of its negatively charged phosphate groups with the positively charged dye molecules.

A rapid procedure for the staining of chromosomes in fixed animal materials.

This paper presents a very simple and reliable procedure for the staining of animal chromosomes employing dyes, such as pyronin G, acridine red, rhodamine B, rhodamine 3GO, belonging to aminoxanthene group and brilliant cresyl blue and methylene violet 3RD, belonging to quinone-imine group. The procedure has been tried on sections of the grasshopper and mouse testes fixed in Dutt's modification of Nawaschin mixture. The method is to deparaffinise sections and then to stain with aqueous solution of these dyes for 2--3 minutes, rinsed with water and dehydrated through grades of ethanol, keeping for 15--30 seconds in each grade with several dips. Preparations are then cleared in xylene and mounted. Stained preparations following this procedure revealed excellent colouration of the chromosomes at all the various stages of mitosis and meiosis, particularly in the case of the grasshopper. Mouse chromosomes stained with these dyes following the same method revealed perfect colouration of the fully condensed chromosomes at all stages of mitosis and meiosis but not of the very early stages, except the sex chromosome. Moreover, grasshopper testis sections when treated with cold concentrated phosphoric acid for varying time-periods and then stained with these dyes also revealed excellent colouration of the chromosomes. The implications of these findings have been discussed.

Effect of cimetidine on renal function in man.

1 Renal function was studied in nine patients with chronic peptic ulcer before and at repeated intervals during treatment with cimetidine (1.6g daily). 2 Plasma creatinine concentration was significantly increased on the first day after starting cimetidine, and at 3 weeks, but not at 12 weeks. Blood urea concentration was unchanged. 3. Clearances of creatinine 51Cr EDTA and 125I-hippuran were significantly reduced within 6 h of starting cimetidine. Clearances of 51Cr EDTA and 125I-hippuran returned to baseline within 3 weeks, and creatinine clearance within 12 weeks. 4 Urinary creatine excretion was significantly increased at 3 weeks, but there was no significant change in urinary creatinine excretion, or in serum creatine phosphokinase concentration. 5. These observations suggest that cimetidine causes an early but short-lived fall in glomerular filtration rate (GFR) and effective renal plasma flow. The later rise in plasma creatinine was unaccompanied by any change in GFR, and may have been due to competition by cimetidine for renal tubular handling. 6 Caution should be exercised when administering cimetidine to patients with pre-existing renal failure.

Influence of UV rays on Feulgen-type staining with azure A-SO2 prepared with normal hydrochloric acid and sodium thiosulphate.

This communication presents a new method for the preparation of azure A-SO2 for use in Feulgen procedure. The salient feature of this method lies in the fact that azure A-SO2 can be decolourised with normal hydrochloric acid and sodium thiosulphate. The pH of this dye reagent is 2.3 and it is of water colour after filtration. The pH of this dye-reagent is raised to 4.0 with an aqueous solution of sodium hydroxide. Nuclear colouration with this newly developed dye-reagent on acid-hydrolysed DNA of tissue sections becomes fairly satisfactory under the usual laboratory conditions. Staining with this dye-reagent under exposure to UV ray is, however, vastly improved within 5 minutes as compared with the control. Stained sections do withstand treatment in SO2 water without exhibiting any leaching of the dye from the nuclei. Possible mode of action of UV rays in increasing the intensity of staining as well as the speed of reaction has been suggested.

Preparation of a new red dye from Janus black and its use in the staining of DNA-aldehyde molecules.

This communication presents a method for the preparation of a new red dye from an aqueous solution of Janus black by adding NHC1 and sodium thiosulphate to it. This new red dye when used on acid-hydrolysed tissue sections reveals the presence of red nuclei when sections after staining are dried between folds of filter paper, differentiated in n-butanol, cleared in xylene and mounted. Similarly stained sections when treated with SO2 water show partial leaching of the dye from the nuclei. Tissue sections when treated with cold concentrated phosphoric acid for 20 min and then stained with an aqueous solution of Janus black reveal the presence of orange-red nuclei. The new red dye obtained from Janus black does not respond to treatment under UV rays. The in vitro absorption data of the red dye indicate peaks at 210, 270 and 545 nm. The in situ absorption spectra of nuclei stained with the new red dye following Feulgen procedure reveal the peak of maximum absorption at 560 nm and those of nuclei treated with cold concentrated phosphoric acid and then stained with this red dye reveal peak at 530--540 nm. Some relevant points raised out of this investigation have been discussed.

Demonstration of DNA with an extra-sensitive Schiff's reagent and a Schiff-type dye-reagent, toluidine blue O-SO2.

This paper describes a method for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2 for use in Feulgen procedure. The method involves replacement of the usual N HCl by N H2SO4 and the usual amount of potassium metabisulphite. Following this method of preparation, an extra-sensitive Schiff's reagent is obtained which requires only 4-5 min for optimum nuclear colouration even when staining is performed at 5 degrees C. This Schiff's reagent produces perfect Feulgen staining up to 6 months after preparation. Toluidine blue O-SO2, prepared with N H2SO4 and potassium metabisulphite, also produces perfect Feulgen type staining of the DNA-aldehyde molecules of acid-hydrolysed mammalian tissue sections. Toluidine blue O-SO2 when shaken with activated charcoal and filtered produces very satisfactory result. The shell-life of this dye-reagent is just a week. The suitability of the use of N H2SO4 for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2, has been discussed.

Propionic acid-metabisulphite-Schiff reagent for quick Feulgen staining.

This communication presents a method for the preparation of Schiff reagent in which N hydrochloric acid has been replaced by a low concentration of propionic acid. The result of using such a Schiff reagent indicates that the dye-reagent thus prepared is extrafast in action on acid-hydrolysed mammalian tissue sections. The intensity of nuclear colouration is also increased considerably, since the pH of the dye-reagent is 6.0. It is, therefore advocated that this newly developed Schiff reagent be used at 5 degrees C.