In vitro determination of active bile acid absorption in small biopsy specimens obtained endoscopically or surgically from the human intestine

BACKGROUND: In the construction of a Kock reservoir for continent urinary diversion, 70 cm of the distal ileum are used. Impaired absorption of bile acids in these patients might cause diarrhoea. Data on the absorption of bile acids in different parts of the human intestine are limited. METHODS: Biopsies were taken during endoscopy from the duodenum, the terminal ileum or the right colon, and during surgery 10, 50, 100 and 150 cm proximally to the ileo-caecal valve using standard endoscopy biopsy forceps. The biopsy specimens were incubated in vitro with radio-labelled taurocholic acid at 37 degrees C for 22 or 45 min The radioactivity was determined using the liquid scintillation technique. RESULTS: A linear increase in the uptake was observed, with increased concentrations of taurocholic acid between 100 and 500 microm in all specimens tested, that represented passive uptake or unspecific binding. The active uptake could be calculated from the intercept of the line representing passive uptake with the ordinate. The active uptake in the terminal ileum was 3-4 times greater than 100 cm proximal to the valve. CONCLUSIONS: The active absorption of bile acids in humans can be determined in small biopsy specimens taken using standard biopsy forceps during endoscopy or surgery. This method is suitable for clinical studies of bile acid absorption. Active uptake of bile acids not only takes place in the very distal part of the ileum but also to a considerable degree 100 cm proximally to the ileo-colonic valve. This should be taken into account when selecting the ileal segment for continent urinary diversion.     

Micromethods for quantitative lipid analysis of human liver needle biopsy specimens

Micromethods are described for rapid quantitative lipid analysis of human liver specimens obtained by percutaneous needle biopsy. Total phospholipid, free fatty acids, triacylglycerol and free and esterified cholesterol were separated by thin layer chromatography and, with the aid of an internal standard, quantitated by specific chemical assays. Individual phospholipids were also separated and quantitated. Fatty acid esters were transmethylated and assayed by gas-liquid chromatography. The results of recovery and reproducibility experiments and lipid values for normal human liver are reported. These methods provide a new approach for investigating the pathogenesis of liver disease and may well prove useful in analysing lipids from biopsies of other tissues.     

Determination of bromsulphthalein-glutathione conjugating enzyme activity in needle biopsy specimens of human liver

The results obtained during a 1-year period with a routine semi-automated assay for human placental lactogen have been examined to assess the most effective quality control parameters. Information derived from the shape and position of the standard curve was not of great value. The most useful quality control techniques were firstly the calculation of a restricted population mean; and secondly the inclusion of two pooled plasmas in each assay run. The pooled plasma results gave valuable information both on individual assay runs, and on long-term changes within the assay.     

Imaging glomeruli in renal biopsy specimens

Glomerular capillary loops are complex vascular filters composed of interdigitating podocytes and fenestrated endothelial cells with an intervening proteoglycan-rich extracellular matrix. This arrangement is crucial to maintaining the filtration barrier but renders the glomerulus difficult to analyze by conventional two-dimensional histochemical techniques. When pathologic lesions distort glomerular architecture, its complex morphology is even more challenging to interpret. Fortunately, recent advances in microscopes and computer software now enable glomerular enthusiasts to dissect this complex structure with finer detail. In this review we explore the application of new methodologies such as two-photon microscopy that optimize three-dimensional, multicolor imaging and single-cell segmentation of glomerular components.     

Spectrophotometric plasma iron determination from fingerpuncture specimens

A simple, sensitive, precise and reproducible micromethod for the determination of plasma iron in a 50-μl sample has been devised by modification of the ICSH reference procedure, which requires a 2-ml sample. The micromethod uses microcuvettes and substitutes ferrozine for the bathophenanthroline chromogen of the ICSH. Comparison of plasma iron values on 47 paired specimens obtained by fingerpuncture and venipuncture determined by the micro- and ICSH-methods respectively gave identical means of 15.8 μmol/l (88 μg/dl); the correlation of the two methods was r = 0.92 (p < 0.0001). The micro-method has been successfully used to measure plasma iron levels in fingerpuncture specimens collected in a field study of 429 children.     

Separation and assay of iron proteins in needle biopsy specimens of human liver.

A micro method has been developed for the separation of the principal classes of iron proteins in needle biopsy specimens of human liver. The iron content of the fractions was determined by automated flameless atomic absorption spectrophotometry in a one step procedure. The levels of total iron, transferrin-, ferritin-, haemprotein- and haemosiderin-iron are reported for control tissue.     

Isolation of micro- and macro-droplet fractions from needle biopsy specimens of human liver and determination of the subcellular distribution of the accumulating liver lipids in alcoholic fatty liver

Needle biopsy specimens of liver were obtained from six control subjects with histologically normal liver and 11 chronic alcoholics with fatty liver. Micro- and macro-lipid droplet fractions were isolated by differential flotation. These fractions, together with the sedimenting membranes, were assayed for cholesterol, cholesteryl ester, phospholipid, free fatty acids and triglyceride. Electron microscopy demonstrated marked differences in the range of lipid droplet sizes in the two fractions and biochemical analysis suggested that the microdroplet lipid corresponded to pre-very low density lipoprotein (VLDL) particles. Studies on biopsies from patients with alcoholic fatty liver showed a 2-3-fold increase in triglyceride in both lipid droplet fractions but most of the accumulating triglyceride was sedimentable and membrane-bound. Needle biopsy specimens from two patients with alcoholic fatty liver were fractionated with a vertical pocket re-orientating rotor. The principal organelles were separated and the subcellular distribution of triglyceride, phospholipid and free cholesterol determined. Triglyceride showed a bimodal distribution to a particulate fraction tentatively located to Golgi particles and to droplet-lipid remaining in the sample layer.     

Determination of H+/K+-ATPase activity in human gastric biopsy specimens.

The aim of our work was to develop a method to determine the the H+/K+-ATPase activity of human gastric biopsy samples. Our method is based on the phosphatase activity and the K+-inducible property of the enzyme. K+-inducible pNPPase activity was determined from homogenated corpus and antrum biopsy samples. H+/K+-ase activity was calculated as the difference between the corpus and antrum K+-inducible pNPPase activities. Quality control measurements were done during 20 successive days from pooled homogenates. The total, between-day and between-run, within-day and within-run coefficients of variations were between 10 and 16%. The healthy mean and reference range of K+-inducible pNPPase activity in the corpus was 95.8 (95% CI: 83.4-108.2 mU/mg protein); in the antrum it was 28.3 (21.6-35.0) mU/mg protein. The calculated H+/K+-ATPase activity was 67.2 (56.9-77.5) mU/mg protein. The measured activities were independent of the age and gender. Summarizing our results we have concluded, that our novel method might be a potential tool to gather data about the functional acid producing capability of human gastric mucosa.     

Chemosensitivity testing of small biopsy specimens

Chemosensitivity testing has usually been limited to tumor specimens of 2–3 g or more. We evaluated the miniaturized, improved nucleic acid precursor incorporation assay (MINI-assay) in 100 small biopsy specimens of 1 g or less. Tumor types included malignant melanomas; sarcomas; and breast, colon, lung, and ovarian carcinomas. Overall, 74/100 were evaluable. The evaluability rate was related to size: 41/48 (85%) 0.7–1.0-g specimens were evaluable, as opposed to 22/35 (64%) 0.4–0.6-g and 11/17 (64%) 0.1–0.3-g specimens. Evaluability was not correlated with tumor types or whether the tumor was from a primary or metastatic site, but was related to yield of viable tumor cells. No viable tumor cells were obtained in 17 specimens, and thus no assay was performed. When 10⁵ cells per well were plated, 49/54 (91%) assays were evaluable, but only 25/29 (86%) were successful when fewer cells were used. Four or more anticancer drugs could be tested in 69/74 (95%) of the evaluable assays. Yielding high evaluability rates and requiring fewer viable cells, the MINI-assay has allowed us to perform chemosensitivity tests on previously unusable biopsy specimens.     

Frequency and spectrum of microorganisms isolated from biopsy specimens in chronic colitis.

In 109 patients with chronic diarrhea colonic biopsies were examined for the presence of specific microorganisms. A positive result was obtained in 48% of patients with ulcerative colitis, 50% with Crohn's disease, 21% with non-specific colitis and 36% with non-specific proctitis. Chlamydiae were found most frequently in all groups. Obligate enteropathogenic bacteria were isolated in only three cases of nonspecific colitis. Of the facultatively enteropathogenic organisms Klebsiella and Pseudomonas aeruginosa were isolated in 31% and 24%, respectively, of patients with ulcerative colitis, in 21% and 7% of patients with Crohn's disease, and in 10% and 6% of patients with non-specific colitis. Whereas chlamydial proctitis is a well-known disease, the results of this study point also to a possible pathogenic role of chlamydiae in the proximal colon. Facultatively enteropathogenic organisms causing acute diarrhea might have aetiologic relevance in some cases of chronic non-specific colitis.     

Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure

A method is presented which allows the investigation of almost the complete mitochondrial content of small human skeletal muscle biopsy specimens. Thorough mechanical disruption with a chopper apparatus results in the release of about 50% of the mitochondrial content. Subsequent treatment of the 600 x g sediment with trypsin releases another 30% of the total mitochondrial population. The biochemical characteristics of the two mitochondrial fractions obtained in these two successive steps have been compared. No obvious differences could be established. The procedure is well suited for biochemical investigation of muscle biopsy specimens from patients suspected of suffering from a mitochondrial myopathy.     

Determination of the superoxide dismutase activity in puncture biopsy specimens of the liver in chronic liver diseases]

Analysis of liver biopsy specimens from patients with chronic active hepatitis has shown reduced superoxide dismutase (SOD) and catalase activities. More profound changes were revealed in the patients with fibrosis, cirrhosis, and primary biliary cirrhosis. Suppressed activities of antioxidative enzymes and dissociation of their systemic interaction were found to be related to the pathologic process severity. Measurement of SOD activity in a biopsy specimen appears to be clinically valuable and may be used for the assessment of liver antioxidative defense in patients with liver conditions.