Determination of creatinine and ultraviolet-absorbing amino acids and organic acids in urine by reversed-phase high-performance liquid chromatography.

A simple and reliable method for the determination of urinary creatinine, amino acids and organic acids was developed. A urine sample was preliminarily separated into an organic acid fraction (including neutral species) and an amino acid fraction by cation-exchange chromatography. Both fractions were analysed by reversed-phase high-performance liquid chromatography, with a phosphoric acid-methanol gradient elution system and ultraviolet detection at 210 nm. Relationships between concentrations and peak heights were linear from 2 to 500 microM for the analytes. Overall recoveries were ca. 100%. The concentrations of creatinine for 37 urine samples, from 20 healthy newborns and from 17 patients with several inherited metabolic disorders, were 2.35 +/- 2.29 mM (ranging between 0.27 and 10.15 mM). The method was applied to the determination of several diagnostically useful metabolites in urine. The concentrations of phenylalanine and phenylacetic acid for five urine samples from patients with phenylketonuria were 347 +/- 177 and 282 +/- 224 microM/mM creatinine, respectively. The concentrations of tyrosine and 4-hydroxyphenyllactic acid in the urine of a patient with tyrosinemia were 112 and 1871 microM/mM creatinine, respectively.


[Surgical treatment of colonic diverticulosis: results of a series of 70 cases].

Results are reported on a series of 70 patients operated upon for colon diverticulosis, surgery being elective in only 23 (32.9%) cases. Global mortality (12.9%), was higher in patients over 70 years of age (P < 0.01) and for emergency cases (17% against 4.3% (NS) after cold surgery). Global morbidity was 20%, the onset of septic complications being influenced (p < 0.01) only by treatment with antiinflammatory drugs. Despite the absence of precise factors predictive of the course of diverticular disease, it should be possible to further improve the very poor prognosis in patients with serious septic complications (Hinchley's stages I to IV) by: prophylactic colectomy in symptomatic diverticulosis, improved evaluation by complementary examinations, notably the scanner, of localized septic complications (stages I and II), with the possible association of guided puncture to avoid the septic areas and to allow performance of a cold colectomy without the need for colostomy. Certain severe septic complications may still develop and these cannot be totally prevented by any therapy. In these cases preference is given to resection of the septic focus using mainly Hartmann's operative techniques. Further studies are needed to evaluate those cases where protected anastomotic resection provides the best results, since in the long term the incidence of re-establishment is higher.


[Abscess of the abdominal wall: a rare presentation of xanthogranulomatous pyelonephritis].

A young psychiatric patient presented with a purulent collection in the lower abdominal wall. Simple drainage failed to heal the abdominal wall abscess, and the presence of a primary septic focus was suspected. Full investigation showed a right renal pelvis calculus with xanthogranulomatous pyelonephritis complicated by a retroperitoneal abscess. Recovery was complete after a nephrectomy. A review of the published literature demonstrated the rarity of this presentation, although even more curious cases have been described, including a urinobronchial fistula, as well as catastrophic cases presenting with a massive haemorrhage. Authors are unanimous that ultrasonography should be the first examination, the scanner being also of primordial value for the diagnosis and demonstration of the retroperitoneal spaces ans fascia. Treatment requires both rapid and radical surgical intervention.


[Synthetic prostheses used in surgery to repair abdominal wall defects (excluding groin hernias). Characteristics, in situ behaviour and applications].

This study is an updating on synthetic prostheses used today to repair incisional hernias, eviscerations and abdominal wall defects due to a severe infection or a parietal excision of malignant tumours. The present prostheses are the fruits of the constantly developing plastic industry. Their characteristics and types (mesh or patch), together with the materials they are made of, are studied taking this development into account. The tissular reactions caused by prostheses are different depending on whether the material they are made of is an absorbable one or not. Published data on animal experimentation are largely used in this part of the study. A significant part is also allocated to the pathobiology of prostheses infection on which is based prevention of risk infection in the surgical practice. A synthetic prostheses has to be chosen depending on its characteristics and the tissular reactions it generates. In a septic environment, only absorbable prostheses can be used without risk. As for incisional hernias, which allows the authors to refer to their own expérience, the choice also depends on both the surgeon's own conception of the treatment and its purpose. The defect recovery after parietal excision of malignant tumours is eased by the use of a synthetic prostheses. In the future, synthetic prostheses will be even more used than today in the repair of abdominal wall. A present, the materials they are made of keep on being developed.


[Study of static transcutaneous oximetry before amputation in patients with arteritis].

Transcutaneous oximetry has been used to define the level of amputation in arteritic patients, with discrepant results. We have studied preoperative statix oximetry in 33 arteritic patients at the Leriche-Fontaine Stage IV, who underwent 36 amputations (thighs = 6, Legs+Symes = 14, transmetatarsal = 7, toes = 9). Oximetry included the measurement, at the level of amputation, of the transcutaneous partial oxygen pressure (tc pO2), of the tissue oxygenation ratio (TOR), preferably with a precordial electrode, of the tc pO2 gain after oxygen inhalation and of the gain ratio with the reference electrode. Two patients died postoperatively, the amputation stump did not heal in another 8 patients. The tc pO2 value was 36.6 +/- 16.2 mm Hg in the healed group and 21.1 +/- 19.9 mm Hg in the non healed group. The TOR respectively was 71.2 +/- 32.7% and 38.6 +/- 29.9% in these two groups. These differences were statistically significant. The difference between the two groups in the measurements made after the oxygenation test was not statistically significant. With a tc pO2 threshold at 26 mm Hg, the sensitivity was 73% and the specificity 75%, the positive predictive value 90%, the negative predictive value 46% and the value of the test 73.5%. The thresholds calculated to be 56% for TOR, 11 mm Hg for the gain and 32% for the gain ratio, did not improve the performances of oximetry. In our study, the tc pO2 was the parameter that best allowed predicting healing was obtained with smaller values than the threshold. Other elements such as the general condition and diabetes have played a role in the prognosis.


Monitoring of benzylpenicillin decomposition in gastric contents by capillary zone electrophoresis.

A method has been developed to follow the decay of the antibiotic penicillin, specifically penicillin G or benzylpenicillin, in the gastric contents of laboratory rats. Purification by centrifugation and DEAE cellulose treatment of the stomach contents (diluted with pH 9 phosphate-borate buffer) was sufficient to allow the quantification of penicillin by capillary zone electrophoresis. An internal standard was used to minimize the injection error. The loss of activity was greater in fasted animals, as expected from the lower pH of their gastric contents, than in fed rats. The in vivo kinetics of the decomposition of the antibiotic was compared to that obtained in water and in hydrochloric acid solutions.


[Endoluminal angioplasty of the superior mesenteric artery in the treatment of intestinal angina].

The authors report about the case of a 69-year-old patient suffering from intestinal angina, in whom an endoluminal angioplasty of the superior mesenteric artery for a stenosis located 3 cm from the ostium allowed suppressing the symptoms. The published series of arterial angioplasty for gastrointestinal care are still rare, but they indicate that angioplasty may be an interesting alternative to or a complement to surgery for patients with intestinal angina.


Immunomagnetic separation of tumor necrosis factor alpha. I. Batch procedure for human temporomandibular fluid.

A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.


Characterization of plasma apolipoproteins by capillary electrophoresis.

The main apolipoproteins of plasma high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were analyzed by capillary electrophoresis. Where possible the results were compared with slab sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Addition of the detergent SDS to the running buffer was essential for separation. Separations were carried out in bare silica and polyacrylamide-coated capillaries. The main apolipoproteins of HDL could be separated in an uncoated capillary filled with borax buffer containing 0.1% SDS. Using the coated capillary, a mixture of HDL and LDL apolipoproteins was resolved in less than 12 min. These preliminary studies indicate that capillary electrophoresis is a promising technique for screening plasma apolipoproteins.


[Damage through surgical risk. A persistent challenge].

No surgical act is innocuous and a complication is not inevitably the result of a mistake. Improved definition of the risk involved should allow a new approach to compensation for damages to be envisaged. Publicly or privately sponsored mutual insurance is worth considering for application of the single European act. Without questioning the concept of professional responsibility, a response should be prepared adapted for damages due to medicosurgical accidents for which a fault cannot be demonstrated. By stimulating enlightened reflection, the present study should allow acceptance of this persistent challenge.


Determination of a new calcium antagonist and its main metabolite in plasma by thermospray liquid chromatography-mass spectrometry.

A highly sensitive thermospray liquid chromatographic-mass spectrometric method has been developed for the simultaneous determination of FRC-8653 (I), a new calcium antagonist, and its main metabolite (M-4) in plasma. A deuterated analogue of I was added to the plasma as the internal standard. After the purification and concentration of the plasma sample on bonded-phase disposable columns, the extract was injected into the thermospray liquid chromatograph and analysed by selected-ion monitoring mass spectrometry. The calibration curves obtained were linear over the concentration range 0.5-100 ng/ml. The limits of quantification are 0.5 ng/ml for I and 1 ng/ml for M-4 in plasma, which are sufficient to evaluate plasma concentrations after oral administration to rats.


New, high-sensitivity high-performance liquid chromatographic method for the determination of acyclovir in human plasma, using fluorometric detection.

A high-performance liquid chromatographic method for the determination of acyclovir in human plasma has been developed. It is the first published chromatographic method capable of determining acyclovir in plasma with sufficient sensitivity and for sufficiently long periods of time following oral administration of a standard dose of acyclovir during pharmacokinetic investigations. Following precipitations of the proteins with perchloric acid, the sample is chromatographed with a strongly acidic mobile phase on a reversed-phase column, and is then subjected to fluorometric detection (excitation 260 nm, emission 375 nm). The determination limit is 6-10 ng/ml human plasma. The calibration is linear in the range 10-12,400 ng/ml plasma, with the coefficients of variation less than 8%. The absolute recovery rate is between 102 and 113%. This method has already been used to analyse several thousand plasma samples.


Determination of cetirizine in serum using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection.

A method using reversed-phase high-performance liquid chromatography with ultraviolet detection for the determination of ceterizine in serum is described. The method is sensitive down to 50 ng/ml (250-microliter loop). Sample preparation involves only serum deproteination with perchloric acid and injection of the centrifuged supernatant. Elution is at pH 2.5 with acetonitrile-methanol-0.05 M phosphate buffer (33:9:58, v/v) on a 25 cm x 4.6 mm I.D. Spherisorb S5 ODS2 column. Detection is at 211 nm, its lambda max. For levels above 300 ng/ml the serum sample size is 100 microliter and a 200-microliter sample is necessary for concentrations less than 300 ng/ml. At the 2 micrograms/ml concentration the intra-assay relative standard deviation is better than 2.2%, whilst the inter-assay deviation is 2.6% over eight samples. At 200 ng/ml the intra-assay relative standard deviation is 6% over seven samples. Detector response is linear from 100 ng/ml to 10 micrograms/ml (100-microliter loop).


Selective determination, in plasma, of artemether and its major metabolite, dihydroartemisinin, by high-performance liquid chromatography with ultraviolet detection.

A sensitive and selective reversed-phase high-performance liquid chromatographic method for the determination of artemether and its major metabolite dihydroartemisinin in plasma has been developed. It involves extraction of plasma with dichloromethane, solid-phase separation of the two analytes and acid decomposition prior to chromatography on a C18 Spherisorb column with a mobile phase of acetonitrile-water (50:50, v/v). Run time is 30 min. The assay satisfies all of the criteria required for use in clinical pharmacokinetic studies.


Immunomagnetic separation of tumor necrosis factor alpha. II. In situ procedure for the human gingival space.

An in situ procedure has been developed for the separation of tumor necrosis factor (TNF) alpha directly from the fluid in the human gingival space. Paramagnetic beads coated with anti-TNF monoclonal antibodies were introduced into the gingival space of the subject with a polypropylene-tipped calibrated delivery system and retrieved using a permanent magnet designed to fit into the space. After retrieval, the amount of immunoadsorbed TNF was quantified using an immunochemical assay called the "cluster assay". The results indicate that following the appropriate preparation of the site, over 95% of the beads could be recovered. With this method we found that 62% of those cavities sampled contained TNF and that the values ranged from 0.10 to 13.0 ng/ml with a mean value of 1.7 ng/ml. A comparison of these values with those obtained from the same space using other methods suggests that the immunomagnetic method was more effective in retrieval of TNF. Because the separation is performed in situ we have named the procedure "chromatobiosis".


Investigation of red blood cell fractionation by gravitational field-flow fractionation.

Gravitational field-flow fractionation is used for the separation of particles according to their sizes in the range 1-100 microns: larger particles elute before smaller ones. This phenomenon can be explained as a result of the steric exclusion of the particles from the vicinity of the channel walls, and/or hydrodynamic effects supposedly associated with the inertia of the liquid. The method was used for the investigation of red blood cells. The dependence of the retention ratio on the flow-rate, sample volume, concentration of blood and relaxation time was studied. Analysis of fifteen individual fractions by Coulter counter and reinjection of three other fractions were studied in order to verify fractionation of red blood cells.


Gas chromatographic-mass spectrometric method for the determination of selenium in biological samples.

The determination of selenium by capillary gas chromatography-mass spectrometry (GC-MS), using an enriched stable isotope 76Se as internal standard, is described. Reference values for selenium in human biological fluids (serum, red blood cells and urine) are reported. With the advent of new compact capillary GC-MS (benchtop) instruments, this method will be very simple and accurate for routine analysis.


Capillary gas chromatographic method for the determination of the thromboxane A2 receptor antagonist S-1452 and its metabolites in human plasma.

A capillary gas chromatographic method for the determination of a newly developed thromboxane A2 antagonist, S-1452, and its metabolites in human plasma has been developed. This nitrogenous compound was detected as the free acid (+)-S-145 using a nitrogen-phosphorus detector. The two metabolites bisnor-(+)-S-145 and tetranor-(+)-145 were also assayed. The three compounds, which all possess carboxylic acid group, were converted into their hexafluoro-2-propyl esters with hexafluoropropan-2-ol-pentafluoropropionic anhydride as reagent. The esterification resulted in a decreased background response from the detector, and hence higher sensitivity and better precision. The glucuronides of the three compounds were also assayed after alkaline hydrolysis. The plasma concentration of the glucuronide of I was more than twice that of the free acid. This method allows determination of the three compounds at more than 2 ng/ml with a precision of 2-6% and an accuracy variation of less than +/- 5%. The analysis was performed within 2 min after injection to assay the sample rapidly from the Phase I test. The method is suitable for pharmacokinetic studies.


Capillary gas chromatographic method for the determination of the thromboxane A2 receptor antagonist S-1452 and its metabolites in human urine.

A capillary gas chromatographic method using a sulphur-specific detector (Hall's electrolytic conductivity detector) was established to determine the thromboxane A2 antagonist S-1452 and its metabolites in human urine. The target species were the free acid (+)-S-145 of the drug and its nine metabolites: the three hydroxyl forms of (+)-S-145 (I, II and III), bis-nor-(+)-S-145 (IV) the hydroxylated forms of IV (V and VI), tetranor-(+)-S-145 (VII) and the hydroxylated forms of VII (VIII and IX). These ten compounds, which have the same sulphur-containing functional group in common, were determined simultaneously. Their conjugated forms, which were assumed to be glucuronides, were also assayed after hydrolysis. The first derivatization was esterification with diazomethane. The second, for the hydroxylated compounds, was trimethylsilylation with bis(trimethylsilyl)trifluoroacetamide. The ten analytes appeared as separate peaks without mutual interference during 5 min. Hall's detector distinguished the ten analytes selectively from the other urinary components, which removed the need for complex clean-up procedures and led to higher sensitivity with a lower noise level. The method is sensitive enough for the assay of substances present at more than 0.1 micrograms/ml of urine. All the compounds could be determined with a high level of precision and accuracy, with 2-5% relative standard deviation and within +/- 5% deviation from the actual value. Day-to-day measurements verified the reproducibility of the method. Recovered substances were quantified by following the time course, and the analytical data together with previously obtained plasma data clarified the metabolism pharmacokinetically.


Identification of metabolites of 4,4'-diaminodiphenylmethane (methylene dianiline) using liquid chromatographic and mass spectrometric techniques.

The in vitro metabolism of 4,4'-diaminodiphenylmethane (methylene dianiline, MDA) was investigated using rabbit liver microsomes. Minimal clean-up of the microsomal incubations was carried out using zinc sulphate precipitation followed by solid-phase extraction on Sep-Pak C18 cartridges. Three metabolites were detected in hepatic microsomal incubations, namely the azodiphenylmethane (azo) azoxydiphenylmethane (azoxy) and 4-nitroso-4'-aminodiphenylmethane (nitroso) compounds. The azo and azoxy metabolites were produced enzymatically whereas the nitroso compound may have been formed via a non-enzymatic process. Reversed-phase high-performance liquid chromatography-plasma spray mass spectrometry was used to initially detect these metabolites. Fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry were utilized to further structurally characterise these compounds. Comparison of mass spectral data obtained from synthesised standards with data obtained on the putative metabolites substantiated the characterisation of these compounds.


Automated high-performance liquid chromatographic determination of chloramphenicol in milk and swine muscle tissue using on-line immunoaffinity sample clean-up.

An on-line high-performance liquid immunoaffinity chromatographic (HPLIAC) system for the direct determination of chloramphenicol in milk and swine muscle tissue is described. The system consisted of a dual-column system in which an HPLIAC column was directly coupled to an RP-8 high-performance liquid chromatographic column. Skimmed and deproteinated milk or aqueous muscle tissue extract was directly injected into the HPLIAC column. After a washing step with phosphate-buffered saline, chloramphenicol was desorbed by a glycine-NaCl buffer (pH 2.8) and directly concentrated on the RP-8 column. Next, chromatography was carried out using acetonitrile-sodium acetate buffer as the mobile phase. Chloramphenicol was detected at 280 nm. Mean recoveries from spiked raw milk were 70 +/- 2% (1-50 micrograms/kg) and from spiked swine muscle tissue 64 +/- 2% (10-50 micrograms/kg). The calibration curves were linear in the range 1-200 micrograms/kg spiking levels. Limits of determination were 1 microgram/kg for milk and 10 micrograms/kg for muscle tissue.


High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6-mercaptopurine: quantitation of red blood cell 6-thioguanine nucleotide, 6-thioinosinic acid and 6-methylmercaptopurine metabolites in a single sample.

A reversed-phase high-performance liquid chromatographic assay was developed to quantify intracellular metabolites of the cytotoxic drug 6-mercaptopurine in the human red blood cell. The 6-thioguanine nucleotides, 6-thioinosinic acid and 6-methylmercaptopurine metabolites are measured in a single sample. A similar assay measures the parent thiopurine compounds. The limit of quantitation of the assay is 0.03, 0.03 and 0.12 nmol per 8 x 10(8) red blood cells for the 6-thioguanine nucleotides, 6-thioinosinic acid and the 6-methylmercaptopurine metabolites, respectively.


High-performance liquid chromatographic method for the simultaneous measurement of remacemide (a novel anticonvulsant and cerebroprotectant) and an active metabolite in human plasma.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of both remacemide (a novel anticonvulsant and cerebroprotectant) and an active, major metabolite in human plasma. After the addition of an internal standard, the analytes were extracted from the plasma by ion-exchange solid-phase extraction and measured by an isocratic HPLC system with ultraviolet detection at 210 nm. The recovery of the analytes was > 90%. The standard curves were linear over the range of quantitation of approximately 10-500 ng/ml for remacemide itself and 15-250 ng/ml for the metabolite. Both intra-day and inter-day accuracy and precision data were excellent. Remacemide and its metabolite were shown to be stable in human plasma for at least a year when stored at -20 degrees C.


High-performance liquid chromatographic method for the determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma.

Determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma involved derivatization of the amino and hydroxyl groups with a ultraviolet-absorbing chromophore followed by extraction into an organic phase. Reversed-phase high-performance liquid chromatography with gradient elution was used for the separation of the analyte from the internal standard (2,3-butanediol). The assay was linear in the range 1.0-1000.0 micrograms/ml of plasma and the coefficient of variation varied between 9.6 and 16.3% whereas the accuracy varied between 90 and 108%. The limit of detection for the assay was 0.282 micrograms/ml. Stability of tris(hydroxymethyl)aminomethane in human plasma frozen at -20 degrees C was studied over a period of three month and the data indicated no significant change.


Determination of trimethylamine and related aliphatic amines in human urine by head-space gas chromatography.

A rapid and simple assay procedure employing head-space gas chromatography has been developed for the routine quantification of volatile methylamines and stable trimethylamine N-oxide present in human urine. This assay will enable the rapid screening of patients and aid the diagnosis of fish odour syndrome.


Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4 degrees C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.


Quantification of carnitine esters by high-performance liquid chromatography. Effect of feeding medium-chain triglycerides on the plasma carnitine ester profile.

A high-performance liquid chromatographic (HPLC) technique was developed using commercially available derivatization reagents and commonly used reversed-phase HPLC column chemistry to analyze plasma samples for their carnitine ester content. The method proved to be sufficiently sensitive to determine changes in the carnitine ester profile in plasma resulting from metabolic disorders or metabolic insults. The method was tested using plasma samples obtained from pigs fed medium-chain triglycerides (MCT) of different chain lengths (four to seven carbons). The MCT feeding was associated with transient increases in plasma carnitine and carnitine esters, and feeding odd-chain MCT (tri-C5 or tri-C7) led to elevated levels of propionylcarnitine in plasma.


Reversed-phase chromatographic method for specific determination of glutathione in cultured malignant cells.

A chromatographic method for the specific determination of glutathione in malignant cell lines is described. The method is based on the ability of glutathione-S-transferase to specifically and quantitatively conjugate glutathione to 1-chloro-2,4-dinitrobenzene and chromatographic quantitation of the resultant conjugate, dinitrophenyl-S-glutathione, by reversed-phase liquid chromatography. The assay can be performed on 20,000 g supernatants of cell homogenates without acid extraction. 2-Mercaptoethanol, a sulfhydryl compound often used as a thiol-protective agent to preserve enzymatic activities of a number of enzymes, did not interfere with glutathione determination by this method. The dinitrophenyl-S-glutathione isolated from either standard glutathione samples or from cell homogenates was shown to be identical to authentic dinitrophenyl-S-glutathione using mass spectrometry. Recovery of glutathione in standard samples by the current method was identical to that determined using 5,5'-dithiobis(2-nitrobenzoic acid). Exogenous glutathione added to supernatants of cell homogenate in the presence or absence of 2-mercaptoethanol was also completely recovered.


Use of eluent containing surfactant for the liquid chromatographic analysis of porphyrins by direct serum injection.

A gradient elution reversed-phase high-performance liquid chromatographic method was developed for the direct serum injection analysis of porphyrins based on the use of eluent containing an anionic surfactant (sodium dodecyl sulfate) at a concentration below the critical micelle concentration to elute the serum proteins at the column void volume. Separation and detection performances were tested with a mixture of porphyrin standards containing uro-, heptacarboxylic-, hexacarboxylic-, pentacarboxylic-, copro-, zinc proto- and mesoporphyrin in a model serum consisting of 50 mg/ml bovine serum albumin. Average limit of detection is 0.06 pmol with a 10-microliter injection volume using fluorimetric excitation at the Soret band of porphyrins. The utility of this method for the direct serum injection analysis of porphyrins in human serum was evaluated by investigating serum samples from individuals suffering from iron-deficiency anemia and breast cancer.


Enantiospecific gas chromatographic-mass spectrometric procedure for the determination of ketoprofen and ibuprofen in synovial fluid and plasma: application to protein binding studies.

A method for the enantiospecific quantitation of two commonly prescribed non-steroidal anti-inflammatory drugs (ketoprofen and ibuprofen) is described. The method involves formation of a mixed anhydride of the drug with ethylchloroformate and subsequent conversion to an amide by reaction with optically active amphetamine. The subsequently formed diastereomers are separated by gas chromatography-mass spectrometry using selected-ion monitoring. The assay is capable of quantifying ketoprofen (2 ng/ml) and ibuprofen (3 ng/ml) enantiomers from a 200-microliters sample of synovial fluid or plasma and is particularly suitable for protein binding studies.


Development of a method for the determination of zopiclone in whole blood.

Mass spectrometry in both electron-impact and positive chemical ionisation modes has been used to elucidate the structures of the decomposition products of zopiclone after gas chromatography. A high-performance liquid chromatographic method has been developed for the determination of zopiclone in whole blood. After selective extraction (butyl chloride) the extracts are chromatographed on Spherisorb ODS-5 (5 microns) using dibasic ammonium phosphate-acetonitrile (40:60). The zopiclone is measured by ultraviolet detection with a limit of quantitation of 4 ng/ml. This method has been successfully applied to the determination of zopiclone in post-mortem blood. Zopiclone levels found in five post-mortem cases are presented.


Determination of Oltipraz in serum by high-performance liquid chromatography with optical absorbance and mass spectrometric detection.

Three methods have been developed for the analysis of Oltipraz in serum. A method suitable for routine use employs spiking with a homologous internal standard, off-line solid-phase extraction, high-performance liquid chromatographic separation, and optical absorbance detection at 450 nm. Method detection limit is about 1 ng/ml. A second method, less susceptible to bias from co-eluting interferences, uses a stable isotope-labeled internal standard, similar extraction and separation, and detection by thermospray mass spectrometry. Method detection limit is about 0.2 ng/ml. A third method was developed which can be used without specially synthesized internal standards. It uses on-line solid-phase extraction, with quantification by comparison with external standards. Method detection limit is about 3 ng/ml. Good agreement was observed between these methods and with similar and different methods run in other laboratories. Calibration curves were linear over the entire range which was investigated, i.e., up to 500 ng/ml. Coefficients of variation were similar for all three methods, being about 5%.


Determination of a new bisphosphonate, YM175, in plasma, urine and bone by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method for the determination of disodium dihydrogen(cycloheptylamino)methylene-bisphosphonate monohydrate (YM175) in plasma, urine and bone is described. Plasma obtained in high-dose animal studies is pretreated by Method A, a simple method using 1 ml of plasma, which is based on deproteinization of plasma followed by coprecipitation of the drug with calcium phosphate and removal of excess calcium ions by AG 50W-X8 resin. Plasma obtained in lower-dose clinical studies is treated by Method B, a more sensitive method using 10 ml of plasma, which is based on solid-phase extraction using a Sep-Pak C18 cartridge coupled with Method A. Urine and bone are treated similarly to Method B. The chromatographic system consists of a mobile phase at pH 11, an alkali-stable column and an electrochemical detector operating in the oxidation mode. The determination limit is 5 ng/ml for Method A and 0.5 ng/ml for Method B in plasma, 1 ng/ml in urine, and 25 ng/g in bone.


Simultaneous high-performance liquid chromatographic determination of salicylates in whole blood, plasma and isolated erythrocytes.

A method using liquid-liquid extraction has been developed for the isolation of acetylsalicylic acid and its metabolites, salicylic, gentisic or possibly salicyluric acids, from whole blood, isolated erythrocytes and plasma. Methylene chloride proved to be the best of the organic solvents tested. For whole blood and isolated erythrocytes it was necessary to carry out haemolysis prior to their extraction. The high-performance liquid chromatographic conditions for the quantitation of acetylsalicylic acid and its metabolites from samples of whole blood, erythrocytes and whole plasma were optimized. Separation was performed using reversed-phase chromatography on Separon SGX C18 and ultraviolet detection at 236 nm. A mixture of methanol-water (80:100, v/v) was the mobile phase, acidified with perchloric acid to pH 2.5.


Enantiospecific high-performance liquid chromatographic assay with fluorescence detection for the monoamine oxidase inhibitor tranylcypromine and its applicability in pharmacokinetic studies.

In order to be able to measure low concentrations of tranylcypromine enantiomers in biological material, chiral fluorescent derivatization and high-performance liquid chromatography (HPLC) were employed. The internal standard S-(+)-amphetamine and borate-sodium hydroxide buffer pH 11 were added to plasma or urine sample aliquots. o-Phthaldialdehyde was used for precolumn derivatization in combination with the chiral mercaptan N-acetylcysteine. HPLC resolution of the diastereoisomeric derivatives was possible on an octadecylsilane column. The mobile phase consisted of sodium phosphate buffer solution pH 6.5, methanol and tetrahydrofuran. The fluorescence of the eluate was monitored at 344/442 nm. The intra-day coefficients of variation were below 10%, the limit of determination was 0.5 ng/ml. The assay was found to be applicable for routine analyses in a preliminary pharmacokinetic study, in which an oral dose of 20 mg racemic tranylcypromine sulfate was administered to three healthy volunteers. The plasma concentrations were generally low, and those of S-(-)-tranylcypromine significantly exceeded those of the R-(+)-enantiomer. Average maximum concentrations were 57.5 and 6.3 ng/ml for S- and R-tranylcypromine, respectively. While S-tranylcypromine was well detectable within the whole study period (8 h), R-tranylcypromine concentrations fell below the detection limit after 4 h in two out of the three studied volunteers.


High-performance liquid chromatographic determination of amitriptyline and its main metabolites using a silica column with reversed-phase eluent. Application in mice.

A method was developed for the assay of amitriptyline, amitriptyline N-oxide, nortriptyline, desmethylnortriptyline and E (trans) and Z (cis) isomers of 10-hydroxyamitriptyline and of 10-hydroxynortriptyline in plasma and brain of animals, using high-performance liquid chromatography with ultraviolet detection (254 nm). Single extraction was performed at pH 10.5 from 0.25 ml of plasma or 1 ml of brain mixture. Chromatographic separations were achieved with a silica column and an aqueous methanol mobile phase containing ammonia. This procedure offers high sensitivity (8-10 ng/ml), high linearity (r > 0.99) and acceptable precision (coefficient of variation < or = 13.3%). The method was used to determine levels of amitriptyline and its major metabolites in mice 30 min after a single intraperitoneal administration of amitriptyline (20 mg/kg).


High-performance liquid chromatography-electrochemical detection of 3-methylhistidine in human urine.

A reversed-phase high-performance liquid chromatographic method is described for the determination of 3-methylhistidine content in human urine using pre-column derivatization with phenylisothiocyanate, isocratic elution with 15 mM sodium acetate-acetonitrile (92:8, v/v) and electrochemical detection. The limit of quantitation was 0.1 pmol. The method has been applied in routine analyses of 3-methylhistidine in both clinical and research work.


Determination of eperisone in human plasma by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method was developed to determine eperisone hydrochloride, 4'-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride, in human plasma over the concentration range 0.2-40 ng/ml. Excellent sensitivity was achieved by selection of a favorable fragment ion, m/z 98, of eperisone and reduction of heat decomposition of eperisone by using a splitless injector and a shortened capillary column. The method described here allows the determination of plasma concentrations as low as 0.2 ng/ml, the concentration attained 6 h after a single oral administration of 50 mg. At eperisone hydrochloride concentrations higher than 0.5 ng/ml, the mean inter-day variation of accuracy of the assay was less than 12%.


Improved method for the determination of trimethadione and its demethylated metabolite, dimethadione, in human serum by gas chromatography.

An improved gas chromatographic method, involving the use of a wide-bore capillary column, for the determination of trimethadione and its only demethylated metabolite, dimethadione, in human serum is described. The results indicate that both substances and the internal standard (maleinimide) were well separated with no tailing peak. The detection limit was 10 ng/ml for trimethadione and 50 ng/ml for dimethadione. This improved method is reliable in terms of sensitivity, selectivity and reproducibility for the simultaneous determination of both compounds in human serum.


Determination by high-performance liquid chromatography of hydroxyurea in human plasma.

An assay using reversed-phase high-performance liquid chromatography with electrochemical detection was developed to measure hydroxyurea in plasma at concentrations suitable for pharmacokinetic studies. The sample preparation is simple, the analysis rapid and assays can be batched. The between-run precision is excellent (coefficient of variation = 2.8-4.5%) and the limit of detection is 0.02 mmol/l. Preliminary studies have shown that the method is suitable for pharmacokinetic studies.


Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine.

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C18 column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform-isopropyl alcohol (9:1, v/v) solution. Levels of 2.5.10(-8) M BE in urine (25 pmol/ml) could be determined.


Redeployment of trigeminal motor axons during metamorphosis.

As a consequence of the degeneration and replacement of the jaw muscle fibers in the leopard frog, Rana pipiens, trigeminal motoneurons innervate different targets before and after metamorphosis. This investigation examined the morphological correlates of the reassignment of trigeminal motoneurons during the initial phases of myofiber turnover. Specifically, silver-cholinesterase histochemistry and electron microscopy were used to 1) identify the fate of motor axons within the neuromuscular junctions (NMJs) applied to degenerating larval myofibers and 2) to determine the origin(s) of the motor axons that innervate the postmetamorphic muscle fibers of the jaw. The results demonstrate that the NMJs are retained on larval myofibers throughout their degeneration and are readily identifiable on the residual larval basal laminae that remain after involution of the sarcoplasm. Light and electron microscopic observations provide evidence that both pre- and post-synaptic elements are present on the degenerating fibers. Furthermore, morphometric analyses indicate that the preponderance (86%) of motor axons supplying adult muscle fibers originates from the larval NMJs. This condition suggests that metamorphic redeployment of trigeminal motoneurons occurs through the resumption of growth at the axon terminal supplying larval muscle rather than through the proximal collateralization of these axons and resorption of larval terminals.


The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.


Distribution of catecholamine-containing nerves on blood vessels of the rat trachea.

This study was performed to determine the distribution of catecholamine-containing sympathetic nerves on blood vessels of the rat trachea. The glyoxylic acid method was used to visualize catecholamine-containing axons in tracheal whole mounts, and silicone vascular casts were used to elucidate the architecture of the vasculature. We also examined the relationship of these axons to the trachea's plexus of cholinergic nerves and ganglia, using tracheal whole mounts stained for acetylcholinesterase activity. We found that most catecholamine-containing axons were associated with arterioles located between cartilaginous rings or in the posterior membrane. In both regions, catecholamine-containing nerves were most abundant at the origin of terminal arterioles, which supplied the airway mucosa and smooth muscle. At the origin of these vessels, the fluorescent axons changed their orientation from longitudinal to circumferential. Few fluorescent axons were present beyond this region of the terminal arterioles, and none was found on capillaries or venules or on smooth muscle cells of the posterior membrane. Fluorescent axons were present in some tracheal ganglia but non enveloped neuronal cell bodies or had varicosities, and no ganglion cells had glyoxylic acid-induced fluorescence. Catecholamine-fluorescence was also present in clusters of small intensely fluorescent (SIF) cells, which were located in the adventitia of the posterior membrane and in the longitudinal nerve trunks which ran the length of the trachea. Pargyline pretreatment increased the fluorescence of axons and SIF cells but did not reveal a different distribution of these structures.(ABSTRACT TRUNCATED AT 250 WORDS)


Distinctive pattern of organisation in the retinofugal pathway of a marsupial: I. Retina and optic nerve.

The nasotemporal division in the retina and the pattern of crossed and uncrossed axons in the optic nerve were determined in an Australian marsupial, a wallaby, Setonix brachyurus (the quokka), following unilateral horseradish peroxidase injections into primary visual centres. The gross morphology of the nerve was also examined. Ipsilaterally projecting ganglion cells were restricted to the temporal retina, whereas those that project contralaterally were located in all retinal regions. The morphological study of the nerve showed that fasciculation patterns, evident along much of the length of the nerve, became indistinct centrally and were replaced in the prechiasmatic region by dorsoventrally oriented fissures. In this prechiasmatic region, axons were oriented in two directions. Whereas the majority were aligned centroperipherally with the long axis of the nerve, a proportion were aligned dorsoventrally in the fissures. Labelling with HRP revealed that uncrossed axons were restricted to the lateral region of the optic nerve and possibly to discrete fascicles, whereas those destined to cross at the chiasm occupied all regions of the nerve but were less dense on the lateral side. This spatial distribution of crossed and uncrossed projections did not change along the length of the nerve. These results demonstrate that fibre organisation in the marsupial optic nerve is different than that found in eutherian mammals.


Distinctive pattern of organisation in the retinofugal pathway of a marsupial: II. Optic chiasm.

In the mammalian optic chiasm retinal axons from each eye divide into two populations, those that decussate and those that remain uncrossed. In eutherian (placental) mammals, the separation of these pathways is not reflected in the structure of the chiasm. The two populations from each eye are mixed through each hemichiasm, segregating only at the midline, where the uncrossed projection turns back. In this study the optic chiasm of a marsupial, the wallaby, Setonix brachyurus (quokka) has been investigated with staining and neuronal tracing techniques. The chiasm of this mammal is quite different from that of eutherian mammals. In coronal section it can be morphologically subdivided into three regions, a central body in which fasciculated groups of axons from each eye interdigitate across the midline, and two distinct lateral regions, one on each side, which contain the uncrossed retinal projections. In the rostral chiasm the lateral regions are separated from the main body of the chiasm by vertically oriented fibre-free regions. Caudally, the lateral regions increase in size and become less distinct as increasing numbers of contralaterally projecting axons that have crossed the midline project into them. However, the two populations remain predominantly segregated in this region. As the lateral regions develop, the central body of the chiasm becomes thinner and finally detaches at the midline to form the two optic tracts. The routes taken by retinal axons through the eutherian and marsupial chiasm appear to be fundamentally different. Therefore, the developmental factors that determine the laterality of retinal projections are likely to show significant differences in the two mammalian groups.


Time-course and extent of retinal ganglion cell death following ablation of the superior colliculus in neonatal rats.

This study has examined the deleterious effect of superior colliculus (SC) ablation on the viability of identified retinotectally projecting ganglion cells in the neonatal rat retina. The time-course and extent of lesion-induced retinal ganglion cell (rgc) death has been determined and an estimate obtained for the rate of clearance of individual dying neurons. In order to demonstrate the projection of rgcs to the SC and the subsequent death of these same neurons after SC lesions, the fluorescent dye diamidino yellow (DY) was injected into the left SC of anesthetized 2 day old Wistar rats (P2: day of birth = P0). DY retrogradely labels the nuclei of tectally projecting rgcs; if these identified rgcs subsequently die, their DY-labelled nuclei become pyknotic and can be visualized in retinal wholemounts. At P4 the rats were again anesthetized and the injected area, seen as a yellow patch in the SC, was removed by aspiration. Rats were perfused 2 to 336 hours after the lesion and retinal wholemounts of the right eye were prepared. Control rats received only DY injections and were perfused at times corresponding to the lesioned animals. In three sham-operated rats; the injected SC was reexposed at P4 but the tectal tissue was not removed. In each of the 42 rats that were analyzed, about 10% of the retina containing retrogradely labelled rgcs was counted; the number of pyknotic versus normally labelled rgcs was determined and changes in normal cell density were also assessed. Pyknotic rates in control and sham-operated rats were similar (average 0.8%, n = 11). In SC-lesioned rats, the proportion of pyknotic DY-labelled rgcs increased to about 2.5% 4 to 8 hours postlesion (PL); the peak period of death occurred at 23 hours PL (8.0%). The amount of pyknosis decreased thereafter and most dying cells had been eliminated by 50 hours PL. Phagocytosis of dying cells was a common feature of retinae in SC lesioned rats. In the long-term (336 hours) rats, counts of normal DY-labelled rgcs in corresponding regions of control and lesioned rats revealed an average decrease in rgc density of 47.3% after P4 tectal ablation. Calculations suggest a clearance time of about 3 hours for dying neonatal rgcs.


Distribution and innervation of lateral line organs in the axolotl.

The lateral line system in axolotls consists of three types of receptors and the cranial nerves that innervate them. Superficial neuromasts, which are mechanoreceptors, are distributed in lines on both the head and trunk. Eight cephalic and three trunk lines can be distinguished on the basis of their innervation and differences in the orientation of the major axes of their neuromasts. A combination of histological techniques reveals that five separate pairs of cranial nerves innervate the neuromasts: anterodorsal lateral line nerves innervate cephalic supraorbital and infraorbital lines; anteroventral lateral line nerves innervate cephalic angular, oral, jugal, and preoperculomandibular lines of the cheek and lower jaw; middle and supratemporal lateral line nerves innervate the cephalic postotic lines; and posterior lateral line nerves innervate the trunk lines. In addition to lines of neuromasts, a second class of mechanoreceptive lateral line organs, pit organs, also occur on the head. Pit organs are smaller than neuromasts but are innervated in a manner identical to that of neuromasts: the sensory hair cells of a single pit organ are innervated by two afferent fibers and a single efferent fiber. Although they occur in localized clusters rather than lines, the four distinct pit organ clusters occurring in axolotls are referred to as the anterior, middle, middle cheek, and gular pit lines as they appear to be homologous to the similarly named pit lines in fishes on the basis of their topography and innervation. In addition to neuromasts and pit organs, ampullary organs comprise a third class of lateral line receptors and are restricted to the head. These electroreceptors occur singly or in small clusters adjacent to the neuromast lines and along the base of the external gills. The ampullary organs adjacent to the supraorbital and infraorbital lines are innervated by the anterodorsal lateral line nerves, whereas all other ampullary organs of the head are innervated by the anteroventral lateral line nerves. Thus postotic ampullary organs and neuromasts of the head are innervated by different lateral line nerves. Examination of the sensory ganglia of the lateral line nerves with respect to the ganglia of the other cranial nerves indicates varying levels of fusion. The ganglia of the anterodorsal lateral line and profundal-trigeminal nerves are totally separate throughout their rostrocaudal extent. The ganglia of the anteroventral lateral line and facial nerves form a fused ganglionic complex with the larger pigmented cells of the anteroventral nerve occupying the dorsal portion of the ganglionic complex.(ABSTRACT TRUNCATED AT 400 WORDS)


Development of GABA-immunoreactivity in the neocortex of the mouse.

The prenatal and postnatal development of GABAergic elements in the neocortex of the mouse was analyzed by GABA-immunocytochemistry. Radial distribution of cells and laminar numerical densities were calculated at each developmental stage to substantiate qualitative observations. The first immunoreactive neurons were observed in the cortical anlage at embryonic day 12-embryonic day 13 (E12-E13) in the primitive plexiform layer. At following prenatal stages (E14-E19), most GABA-positive neurons were present in the marginal zone, subplate, and subventricular zone. GABA-immunoreactivity in the cortical plate appeared early (E14), although the complete maturation of its derivatives was achieved postnatally. At prenatal stages we noted a well-developed system of immunopositive fibers in the subplate. As indicated by the direction of growth cones, most of these fibers had an extracortical origin and invaded the cortex laterally through the internal capsule and striatum. In rostral and middle telencephalic levels, fibers originating in the septal region contributed to the cingulate bundle. Presumably corticofugal fibers and callosal axons were also noticed. At postnatal stages the maturation of GABA-immunoreactivity appeared to be a complex, long-lasting process, in which the adult pattern was produced at the same time as the appearance of certain regressive phenomena. Thus, between postnatal day 0 and postnatal day 8 (P0-P8), GABA-positive populations disappeared from the subventricular zone, marginal zone and to a lesser extent from the subplate. At the same ages we noticed the presence of morphologically abnormal, GABA-immunoreactive neurons in the subventricular zone and subplate which are interpreted as correlates of neuronal degeneration. Most GABA-positive subplate fibers also disappeared whereas GABA-immunoreactive axons were seen in the cingulate bundle until the adult stage. In the derivatives of the cortical plate, the maturation of GABA-immunoreactive elements progressed according to the "inside-out" gradient of cortical development, with the important exception of layer IV, which was the last layer to exhibit an adult-like appearance. Within each layer deriving from the cortical plate (layers VIa to II-III), GABA-immunoreactivity showed a protracted maturation in which the first GABA-positive cells were detected a few days after cell birth but substantial numbers of neurons began to express GABA considerably later. The later phase occurred concurrently with the maturation of GABA-positive axonal plexuses. These results suggest that different GABA-positive populations show different developmental regulation of GABA expression during cortical ontogenesis.


A quantitative study of the human cerebellum with unbiased stereological techniques.

Estimates of total number of different neuron and glial cell types in the dentate nuclei and the four major regions of the human cerebellum were obtained by new stereological methods. With the optical disector and Cavalieri methods, the total number of neurons and glial cells in the human cerebellum of five elderly men was estimated to be 105,000 x 10(6) (coefficient of variation, CV = 0.13). Of this total, the granule cells comprised 101,000 x 10(6) (CV = 0.13) and the Purkinje cells comprised 30.5 x 10(6) (CV = 0.13) of the neurons. The mean of the total number of neurons in the dentate nucleus was 5.01 x 10(6) (CV = 0.28). The average surface area of the human cerebellum was estimated to be 1160 cm2 (CV = 0.29). The rationale for the different sampling schemes used to quantify the various cell types is described.


Effects of neonatal transection of the infraorbital nerve upon the structural and functional organization of the ventral posteromedial nucleus in the rat.

The present study examined the way in which an indirect partial deafferentation of the medial portion of the ventrobasal complex (VPM/VPL) induced by neonatal transection of the infraorbital nerve (ION) altered the structural and functional properties of its constituent neurons. This manipulation significantly reduced the volume of the contralateral VPM/VPL. In addition, cell counts in Nissl-stained material revealed a significant reduction of the number of VPM/VPL neurons contralateral to neonatal ION transection. We also analyzed the effect of neonatal ION transection on the soma-dendritic morphology of individual neurons in the ventral posteromedial nucleus of the thalamus (VPM) by intracellular injection of horseradish peroxidase (HRP) in vivo and Lucifer yellow in fixed slices. Neonatal transection of the ION resulted in increased dendritic length, area, and volume of VPM neurons in both preparations; however only the changes observed in fixed slices reached statistical significance. Alterations in the functional characteristics of VPM neurons were also observed following neonatal nerve damage. There was a significant decrease in the percentage of vibrissae-sensitive neurons and a corresponding increase in the percentages of neurons responsive to guard hair deflection or that were unresponsive to peripheral stimulation. Neonatal nerve damage also resulted in significantly longer latencies of VPM cells after stimulation of either trigeminal nucleus principalis or subnucleus interpolaris. The present results indicate that the development of normal response properties and soma-dendritic morphology of VPM neurons is dependent upon intact afferent input during development. Indirect partial deafferentation of VPM/VPL by neonatal transection of the ION results in reduced neuron number, which may result in decreased competition among the dendrites of these neurons. This proposal is consistent with observations of increased dendritic dimensions of VPM neurons contralateral to neonatal ION damage.


PHA-L analysis of projections from the supramammillary nucleus in the rat.

The projections of the supramammillary nucleus (SUM) were examined in the rat by the anterograde anatomical tracer Phaseolus vulgaris leucoagglutinin (PHA-L). The majority of labeled fibers from SUM ascended through the forebrain within the medial forebrain bundle. SUM fibers were found to terminate heavily in the hippocampal formation, specifically within the granule cell layer and immediately adjoining molecular layer of the dentate gyrus. In addition, SUM fibers were shown to distribute densely to several structures with strong connections with the hippocampus, namely, the nucleus reunions of the thalamus, the medial and lateral septum, the entorhinal cortex, and the endopiriform nucleus. SUM fibers were also shown to project significantly to several additional subcortical and cortical sites. The subcortical sites were the dorsal raphe nucleus, the midbrain central gray, the fields of Forel/zona incerta, the dorsomedial hypothalamic area, midline/intralaminar nuclei of the thalamus (posterior paraventricular, rhomboid, central medial, intermediodorsal, and mediodorsal), the medial and lateral preoptic areas, the bed nucleus of the stria terminalis, the substantia innominata, the vertical limb of the diagonal band nucleus, and the claustrum. The cortical sites were the occipital, temporal, parietal, and frontal cortices. Some notable differences were observed in projections from the lateral as compared to the medial SUM. For example, fibers originating from the lateral SUM distributed heavily to the hippocampal formation and parts of the cortex, whereas those from the medial SUM projected sparsely to these two regions. The SUM projections to the hippocampal formation and associated structures may serve as the substrate for a SUM involvement in the generation of the theta rhythm of the hippocampus and the gating of information flow through the hippocampal formation.


Translaminar deficits in the retinae of albinos.

The central retina in albino mammals is poorly developed. There is a general reduction in ganglion cell density compared with the normal animal, and there are irregularities in the center to periphery gradient in ganglion cell density. It is not known whether, and if so to what extent, this abnormality is associated with deficits in other retinal layers. In this study the distribution of cells in the ganglion cell layer has been determined in horizontally sectioned eyes from pigmented and albino ferrets. This was undertaken to define the location of the region of highest cell density and the relative cell gradients around it. Detailed counts and measurements were then undertaken to determine the cell density within, and thickness of, the inner and the outer nuclear layers in these animals. All the albino animals had an abnormal distribution of cells in the ganglion cell layer in the central retina when compared with pigmented animals. The extent of this abnormality was variable. No differences between pigmented and albino animals could be found in the packing density of cells within the inner or the outer nuclear layer. However, in the pigmented animals there was a clear increase in the thickness of these layers associated with the region of highest density in the ganglion cell layer. This feature was absent in the albino animals, where the gradient in layer thickness was less marked and frequently contained irregularities. These abnormalities were most obvious in the outer nuclear layer.


Assessing environmental health concerns associated with nitrous oxide.

Concerns about the effects of occupational exposure to nitrous oxide have existed for a decade. Unfortunately, data are lacking that document biologically tolerable exposure levels. Current guidelines regarding use of N2O are reviewed.


A year of chronic TMPDS: relating patient symptoms and pain intensity.

Pain intensity among 136 women with TMPDS was compared with the signs and symptoms of TMPDS. Most masticatory muscle pain was related to TMPDS pain intensity. Clicking, restricted mandibular range and evidence of tooth grinding were not related to pain intensity.


Endodontic case selection: to treat or to refer.

Endodontic treatment in the United States is delivered primarily by general dentists. A dilemma often arises as to which cases should be treated and which should be referred for specialty care. The case selection system described here rates endodontic cases so that practitioners can assess those cases they are best qualified to treat.


How dentists see themselves, their profession, the public.

The public traditionally has held dentistry in high regard as a profession. But some recent evidence suggests a decline in public confidence--a fact that may be affecting the way dentists view themselves. What should and can be done?


Low early gallstone recurrence rate after successful extracorporeal lithotripsy in patients with solitary stones.

Piezoelectric lithotripsy with the EDAP LT-01 machine combined with adjuvant bile acid therapy results in complete clearance of radiolucent gallstones in selected patients. We assessed stone recurrence rate in 84 patients with complete clearance of stone fragments and followed up at least 12 months after cessation of bile acid therapy (mean 17 months, range 12-33). Fifty-four patients had a solitary stone and 30 multiple stones. Bile acid therapy was continued for 3 months after complete fragment stones clearance which was ascertained by two consecutive ultrasound examinations. Stone recurrence was assessed by ultrasonography at 6 and 12 months, and then at least once a year. Gallstone recurrence occurred in 5 patients (6%) between 9 and 12 months with no further recurrence up to 33 months. The rate of recurrence at one year was 3.7% in patients with a solitary stone and 10% in patients with multiple stones. Only one patient with stone recurrence had recurrent biliary pain. We concluded that early gallstone recurrence rate after successful lithotripsy seems to be low in patients with solitary stones.


Endocytosis of soluble IgG immune complex and its transport to lysosomes in hepatic sinusoidal endothelial cells.

Both Kupffer cells and sinusoidal endothelial cells are engaged in the hepatic uptake of soluble IgG immune complex (IgG-IC) through Fc-receptors on their surface. Hepatocytes have also been reported to take up IgG-IC. It remains unclear, however, whether the endothelial cell degrades IgG-IC and whether the hepatocyte participates in IgG-IC clearance. In this study, normal mice received a single intravenous injection of soluble immune complex preformed in antigen excess, i.e. bovine serum albumin (BSA) anti-BSA-mouse-IgG complex (BABIgG) or BSA anti-BSA-mouse-F(ab')2 complex (BABF(ab')2), or BSA alone. An immunoperoxidase study for BSA showed that from 1 to 120 min after injection only BABIgG was ingested by both endothelial cells and Kupffer cells but not by hepatocytes. The staining intensity of BABIgG was maximal at about 15 min and decreased subsequently. Endocytosis of BABIgG occurred through coated pits in the endothelial cells. Within a few minutes, endocytosed BABIgG was found in tubulovesicular structures and large vesicles. The occasional large vesicles were shown to be lysosomes by simultaneous demonstration of BABIgG with acid phosphatase. BABIgG was not found on either of the endothelial and hepatocellular surfaces facing the space of Disse or in hepatocytes. These results indicate that soluble IgG-IC is endocytosed by sinusoidal endothelial cells and degraded in the lysosomes and that the participation of hepatocytes in the clearance of IgG-IC is improbable.


Membrane-bound phenylalanine hydroxylase of human liver.

Phenylalanine hydroxylase (EC 1.14.16.1) antigen and activity have been identified among proteins extracted with a buffer containing 0.4% Triton X-100 from adult human liver bioptate fraction, which was sedimented at 105,000 x g (n = 4). This enzyme fraction was designated as a 'membrane-bound form of phenylalanine hydroxylase'. It amounted to 5-15% of phenylalanine hydroxylase activity and 15-25% of phenylalanine hydroxylase antigen content. After immunoblotting two-dimensional gels, the soluble (cytoplasmic) form of phenylalanine hydroxylase antigen displayed three spots: one spot corresponded to the L-subunit with a molecular weight of 55,000, the two other spots corresponded to the H-subunit with a molecular weight of 57,000. Only the L-subunit was revealed in the membrane-bound enzyme form. Both phenylalanine hydroxylase activity and antigen were also demonstrated in extracts from human embryonic livers (n = 7). However, in this case the membrane-bound phenylalanine hydroxylase amounted to 85% of the antigen content. Subunit compositions of the enzymes were similar in adult and embryonic livers. The differences in the subunit compositions and enzyme activities of membrane-bound and cytoplasmic forms of phenylalanine hydroxylase in adults and embryos may be due to other functions of this enzyme in the hepatocyte membrane.


The recognition site for hepatic clearance of plasma kallikrein is on its heavy chain and is latent on prokallikrein.

We partially purified the glycoproteins prokallikrein and kallikrein from rat plasma. The purification of rat plasma kallikrein may result in two forms: an intact form (alpha, M(r) 84-87 kDa) and a partially degraded form (beta, M(r) 46-51 kDa). The alpha-form is composed of a heavy chain (M(r) 50 kDa) and a light chain (M(r) 34-37 kDa) linked by a disulfide bond. The catalytic site is found on the light chain. The beta-form has a partially degraded heavy chain (M(r) 28 kDa). Using a preparation of exsanguinated and perfused rat liver, we verified that rat plasma prokallikrein is not activated by the liver and that neither the proenzyme nor the light chain is removed by the organ. Both forms (alpha and beta) of the active enzyme are similarly removed from the perfusate. We also observed that the clearance of plasma kallikrein is temperature-dependent, and not affected by substances that inhibit binding to galactosyl-, mannosyl-, fucosyl- or phosphomannosyl-specific lectins, but inhibited by beta-galactosides. We suggest that: (a) the binding site to hepatocytes is latent on prokallikrein and is located on its heavy chain, more specifically on the 28-kDa fragment still present in the beta form of the active enzyme and (b) plasma kallikrein is recognized by an S-type lectin.


Liver fibrosis in genetic hemochromatosis. Respective roles of iron and non-iron-related factors in 127 homozygous patients.

A retrospective study of 127 patients with untreated homozygous genetic hemochromatosis (HGH) was conducted to evaluate the respective roles of iron overload and non-iron-related factors in the development of hepatic fibrosis in HGH. Twenty-seven percent of the patients had cirrhosis, 21% had liver fibrosis and 52% had no fibrosis (prefibrotic group). The mean value of liver iron concentration was increased significantly (p < 0.001) in cirrhotic (378 +/- 144 mumol/g dry wt.) and in fibrotic (331 +/- 168) subjects compared to prefibrotic (237 +/- 108) patients. Of 13 patients with liver iron concentration > or = 500, 12 had liver fibrosis or cirrhosis, versus 48/134 with liver iron concentration < 500. Chronic alcoholic men exhibited hepatic fibrosis or cirrhosis more frequently than non-alcoholic men (p < 0.001). Non-alcoholic men had hepatic fibrosis or cirrhosis more often than non-alcoholic women (p < 0.05). Cirrhotic and fibrotic patients were significantly older than prefibrotic patients whilst a significant correlation between age and liver iron concentration was found in younger patients only. These results suggest that the iron overload threshold necessary to induce fibrosis is modulated by non-iron-related factors such as alcoholism, sex and age. The development of fibrosis in HGH with liver iron concentration < 500 mumol/g is frequent and must lead to a search for associated non-iron-related fibrogenic factors.


Thermogenic response to intravenous nutrition in patients with cirrhosis.

Energy expenditure was determined using continuous indirect calorimetry in the basal state and during 3 h of total parenteral nutrition (TPN) in 8 patients with cirrhosis and 8 healthy volunteers. TPN consisted of glucose, fat and amino acids and had a glucose/fat ratio of 50:50. The infusion rate was set to provide energy corresponding to 62.5% of the individually measured 24-h resting energy expenditure. In the basal state energy expenditure was similar in patients and controls while the respiratory quotient (RQ) was lower in the patients (0.78 +/- 0.01 vs. 0.82 +/- 0.01, mean +/- SEM, p < 0.05). During TPN, energy expenditure increased progressively during the 3-h infusion period. The average rise in energy expenditure was similar in patients (19.1 +/- 1.2%) and in controls (21.4 +/- 1.6%, n.s.). The RQ rose in both groups, but more in the patients with cirrhosis. At the end of the study, RQ was higher in patients (0.89 +/- 0.01) than in controls (0.85 +/- 0.01, p < 0.05). It is concluded that the nutrient-induced rise in energy expenditure during TPN is not significantly different in patients with cirrhosis and control subjects. Furthermore, the results indicate that the increased fat utilization in overnight fasting cirrhotic patients is rapidly shifted to an augmented carbohydrate oxidation during TPN, possibly as a consequence of marked hyperinsulinemia.


Intrahepatic expression of hepatitis B core and surface antigens in chronic hepatitis delta-virus infection.

To evaluate the interference of hepatitis B virus (HBV) protein expression in the liver in chronic hepatitis delta-virus (HDV) infection, the intrahepatic expression of hepatitis B core and surface antigens (HBcAg and HBsAg) was studied in 36 HBsAg carriers who were seropositive for anti-HDV and in 36 anti-HDV negative controls [18 with serum hepatitis B e-antigen (HBeAg) and 18 with anti-HBe]. Of 18 HBeAg-positive patients with anti-HDV, 12 had HDV antigen (HDAg) in the liver. HBcAg was positive in 66.7% (8/12) of the HbeAg-positive patients with HDAg in the liver, and in 94.4% (17/18) of controls (p = 0.14). The distribution of HBcAg was exclusively cytoplasmic in 75% (6/8) of HDV-infected patients, but was mixed nuclear and cytoplasmic in 70.6% (12/17) of the controls. The prevalence and quantitative expression of HBcAg in the nucleus, but not in the cytoplasm, were significantly decreased in chronic HDV infection. HBsAg was positive in 91.6% (11/12) of HBeAg-positive patients with HDV infection and in all controls. Membranous expression of HBsAg was detected less frequently in HDV-infected patients than in controls (7/12 vs. 17/18, p = 0.05), while the prevalence and quantitative expression of HBsAg in the cytoplasm showed little or no difference. HDAg was detected in all of the anti-HBe-positive patients with anti-HDV. Of these, none had HBcAg detectable in the liver, nor did controls, while HBsAg was detected exclusively in the cytoplasm in 94.4% (17/18). The prevalence and quantitative expression of HBsAg in the cytoplasm was not different for HDV-infected patients or controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Polyamine plasma levels and liver regeneration following partial hepatic resection in man.

Polyamines (putrescine, spermidine, and spermine) are widely distributed in animal and vegetal tissues, where their intracellular concentration strictly correlates with normal and pathological cell growth and protein synthesis. By means of a sensitive HPLC technique, the fasting plasma concentrations of polyamines were measured serially in 11 patients who underwent partial hepatic resection because of focal liver lesions. Samples were obtained before surgery and over the next 6 months, during hepatic regeneration. Liver volume was also measured by ultrasound on the basis of the 3 maximum diameters of the liver. From 2 to 4 weeks after surgery, plasma putrescine increased by a maximum of 78%, and spermidine by approximately 50%. No changes were observed in spermine levels. The spermidine/spermine ratio nearly doubled during liver regeneration. The volume of the liver decrease from 1505 [SD 236] ml to 743 [151] ml after resection, and returned to nearly normal values after 6 months (1231 [100] ml, p < 0.05 vs. basal values). The liver regeneration rate was highest 2-4 weeks after resection, and declined thereafter, when prevailing polyamine concentrations returned to normal. These data show that liver regeneration is accompanied by a significant increase in fasting putrescine and spermidine concentrations, which might be biochemical signals of active liver cell regeneration.


Thrombocytopenia post liver transplantation. Correlations with pre-operative platelet count, blood transfusion requirements, allograft function and outcome.

This study reports that thrombocytopenia is a universal phenomenon post hepatic transplantation. In 53 consecutive adult patients undergoing liver transplantation the platelet count fell by a mean of 63% (157 x 10(9)/l to 50 x 10(9)/l). The platelet count reached a nadir at Day 5 post-transplant but returned to pre-operative levels by Day 14. Non-parametric regression analysis found that pre-operative platelet count, blood transfusion requirements and maximum post-operative ALT values were independent predictors of the percentage fall in platelet count. No correlation was seen with length of graft cold ischaemic time or the use of University of Wisconsin (UW) solution. The nadir day correlated with maximum post-operative bilirubin and ALT, graft ischaemic time and use of UW solution. Maximum post-operative ALT was also an independent predictor of nadir platelet count. It was observed that patients who did not survive the hospital admission had lower post-operative platelet counts and these did not return to pre-operative levels by Day 14. The percentage fall in platelet count was an independent predictor of survival. Severe thrombocytopenia was associated with cerebral haemorrhage in 3 patients. This report provides evidence that allograft dysfunction (maximum post-operative bilirubin and/or AST/ALT) was the most consistent independent predictor of the nadir platelet count, nadir day and percentage fall in platelet count post liver transplantation although the exact mechanism(s) of the platelet changes remain uncertain.


In vitro secretion of specific antimitochondrial antibodies in primary biliary cirrhosis.

Antimitochondrial antibodies are present in the serum of virtually all patients with primary biliary cirrhosis. They have a well-defined antigen reactivity that is diagnostic for the disease. The role of these autoantibodies in the disease process remains to be defined. In this study we show that antimitochondrial antibodies can be produced in vitro by peripheral blood lymphocytes, that the cells producing antimitochondrial antibodies are present in the peripheral blood in a high frequency and seem to be maximally activated. Stimulation with pokeweed mitogen did not augment the in vitro production of antimitochondrial antibodies in patients nor did it induce the production of these antibodies by control lymphocytes. Thus, antimitochondrial antibodies are not simply an expression of polyclonal B-cell stimulation. The high frequency of maximally activated B-cells producing antimitochondrial antibodies suggests active antigenic stimulation.


Bed-rest-induced hypernatriuresis in cirrhotic patients without ascites: does it contribute to maintain 'compensation'?

Renal function, plasma renin activity, plasma aldosterone concentration and urine excretion of free norepinephrine were evaluated in 13 cirrhotics without previous or ongoing ascites and in 13 healthy subjects, after 6 days of controlled electrolyte intake (40 mmol of Na and 70 mmol of K per day) and during 24 h of recumbency. Plasma concentrations of the atrial natriuretic peptide (ANP) were also measured in 8 patients and 8 controls. Despite a low-normal filtered load of sodium (14.6 +/- 1.2 vs. 17.1 +/- 1.2 mmol/min), cirrhotic patients showed supernormal natriuresis (141.5 +/- 14.1 vs. 78.8 +/- 8.6 mmol/day; p < 0.001). Whereas the fractional excretion of sodium in these patients was twice that of controls (0.70 +/- 0.05 vs. 0.36 +/- 0.04%; p < 0.001), potassium excretion (42.5 +/- 2.7 vs. 43.1 +/- 2.7 mmol/day) and urine volume (1270 +/- 98 vs. 1452 +/- 148 ml/day) did not differ. In cirrhotics, plasma renin activity was reduced (0.50 +/- 0.12 vs. 1.39 +/- 0.33 ng/ml/h; p < 0.02), and plasma aldosterone concentration tended to be lower (66 +/- 10 vs. 86 +/- 9 pg/ml; p = 0.09), while urine norepinephrine excretion did not significantly differ from controls (961 +/- 120 vs. 782 +/- 43 ng/h). ANP was higher in patients than in controls (92 +/- 17 vs. 48 +/- 9 pg/ml; p < 0.05). Natriuresis was directly correlated with ANP (r = 0.69, p < 0.005) and ANP/plasma aldosterone ratio (r = 0.63; p < 0.01) in patients and healthy subjects taken together.(ABSTRACT TRUNCATED AT 250 WORDS)


Memory T-lymphocytes are the main population of tumor-infiltrating lymphocytes obtained from human primary liver tumors.

The phenotypic characteristics of freshly isolated tumor-infiltrating lymphocytes (TIL) obtained from human liver tumors were analyzed by two-color flow cytometry. TIL consisted of mainly CD3+ T-lymphocytes (70-80%). The ratio of CD4/CD8 in TIL from primary and metastatic liver tumors and autologous peripheral blood lymphocytes (A-PBL) was 1.3, 1.1 and 1.3, respectively. The majority of CD3+ T-lymphocytes (mean +/- SD; 95 +/- 11%) expressed T-cell antigen receptor (TCR) alpha/beta, and gamma/delta TCR positive T-cells were only 5 +/- 4.5% in TIL from both primary and metastatic liver tumors. TIL showed significantly higher percentages of transient activation markers, such as CD25 (Tac) and HLA-DR, than A-PBL. TIL also contained significantly more populations of CD3+ CD45RO+ T-lymphocytes, which are considered to be expressed on primed (memory) T-lymphocytes, than A-PBL. Furthermore, TIL from primary liver tumors demonstrated significantly higher percentages of CD3+ CD45RO+ T-cells than those from metastatic liver tumors. These data indicate that TIL from human liver tumors are an apparently distinct population from A-PBL, and that local immune responses against human primary and metastatic liver tumors might be different. Moreover, TIL from primary liver tumors consisted of mainly activated or primed (memory) T-cells, suggesting that they were sensitized and activated by autologous tumor cells in vivo. These observations may imply the possibility of adoptive immunotherapy using TIL against human primary liver tumors.


Liver transplantation for erythropoietic protoporphyria. Report of a new case with subsequent medium-term follow-up.

We report a new case of successful liver transplantation in a 36-year-old patient with terminal hepatic failure due to erythropoietic protoporphyria. Data regarding protoporphyrin levels in erythrocytes and feces, before and after transplantation, seem to indicate that in this case protoporphyrin overproduction was in part due to liver synthesis. Four years after surgery, the patient is completely free of skin photosensitivity. His liver function tests are normal; there are no visible protoporphyrin deposits or ultrastructural abnormalities in his new liver. However, recurrence of the disease in the long term cannot be excluded, since erythrocyte protoporphyrin levels remained elevated after liver transplantation.


Blockade of ATP-sensitive K+ channels by glibenclamide reduces portal pressure and hyperkinetic circulation in portal hypertensive rats.

Certain results of in vitro studies raise the possibility that blockade of ATP-sensitive K+ channels by glibenclamide may induce vasoconstriction. Therefore, this substance might decrease portal pressure and hyperkinetic circulation in animals with portal hypertension. Thus, systemic and regional hemodynamics (radioactive microspheres) were measured before and 20 min after a bolus intravenous injection of glibenclamide (20 mg/kg) in conscious rats with portal vein stenosis. Blood pressure decreased significantly from 14.5 +/- 1.5 to 12.2 +/- 1.2 (mean +/- SE). Cardiac index significantly decreased by 24%, portal tributary blood flow by 31%, and hepatic artery blood flow by 35%. Systemic vascular resistance significantly increased by 38%, portal territory vascular resistance and hepatic artery vascular resistance by 61%, each, and renal vascular resistance by 17%. Arterial pressure, heart rate, and renal blood flow were unchanged. Moreover, glibenclamide blunted the vasodilating action of diazoxide (an ATP-sensitive K+ channel opener). These results show that in rats with extrahepatic portal hypertension the blockade of ATP-sensitive K+ channels by glibenclamide reduces portal pressure and hyperkinetic circulation.


Comparison of pre-core/core hepatitis B virus region in liver tissue and serum from patients with chronic hepatitis B infection.

We examined the relationship between HBV-DNA isolated from the liver and from the serum in patients with various serological characteristics of chronic hepatitis B infection. Amplification and direct sequencing of the HBV pre-core/core region was carried out in 9 patients who were seropositive for HBsAg and HBV-DNA--4 HBeAg positive and 5 anti-HBe positive. Complete sequence identity was observed between HBV-DNA isolated from the serum and the liver in individual patients. In addition, shortened forms of the HBV core ORF were detected in patients with chronic active hepatitis, but not in patients with chronic persistent hepatitis.


Can hepatocytes serve as 'activated' immunomodulating cells in the immune response?

During the last few years, there has been a growing evidence that hepatocytes are not merely 'passive' target cells for immunological attack by effector T-cells, but may play a more 'active' role in the initiation and perpetuation of the immune response. Immune modulators released by inflammatory cells at the site of inflammation, as well as the eliciting antigen itself, are able to modulate the phenotype of hepatocytes. This would result in abnormal cytokine production and/or cytokine/receptor expression, as well as active synthesis and display of surface immune 'activation' markers and adhesion molecules, which act as co-stimulatory signals for T-cell activation. These accessory functions involve multiple molecular pathways of cell-cell interactions, which in turn will enable hepatocytes to play a role as 'accessory' cells in both the afferent and efferent arms of the cell-mediated immune response.


Diurnal variation in cholesterol metastability of hepatic bile and its acute modulation with ursodeoxycholic acid in humans.

We studied the alteration of cholesterol metastability of hepatic bile caused by diurnal variations in hepatic biliary lipid excretions and acutely induced changes following ursodeoxycholic acid (UCDA) administration. Hepatic bile was collected at 6-h intervals for 24 h from 6 patients with an indwelling choledochal drainage before and after UDCA administration. A basal diurnal variation showed the highest cholesterol saturation index (p < 0.05) and cholesterol distribution in vesicles (p < 0.01) and the shortest nucleation time (p < 0.05) in the early morning. After the ingestion of ursodeoxycholic acid for 1 day, early morning biliary cholesterol concentrations were reduced. Interestingly, significant decreases in vesicular cholesterol concentrations (1.0 +/- 0.2 to 0.1 +/- 0.04 mM, p < 0.01) and in the vesicular cholesterol/phospholipid ratio (1.6 +/- 0.1 to 0.7 +/- 0.1, p < 0.05) were associated with prolongation of the nucleation time (11.5 +/- 1.2 to 18.7 +/- 1.5 days, p < 0.01). Biliary protein had no diurnal variations and did not decrease significantly with UCDA. These results indicate that during a day the early morning hepatic bile is the most unstable and that UCDA acutely enhances hepatic biliary metastability mainly by decreasing the rate of vesicular cholesterol saturation.


'Albumin-mediated transport phenomenon' observed for ligands with high membrane permeability. Effect of the unstirred water layer in the Disse's space of rat liver.

In this paper, we offer experimental evidence of the rate-limiting diffusion of ligands through the unstirred water layer (UWL) as an explanation for the so-called albumin-mediated transport phenomenon. The relative membrane permeability of various ligands was first evaluated using isolated rat hepatocytes. Then, the effect of albumin on the uptake of ligands of a wide range of membrane permeabilities was examined using the perfused rat liver. The results were similar to those expected from the UWL model: ligands with high membrane permeability (warfarin, diazepam and taurocholate) clearly exhibited albumin-mediated transport, those with medium membrane permeability (tolbutamide and salicylate) showed less albumin-mediated transport, and ligand with low membrane permeability (cefodizime) did not show albumin-mediated transport. These results were explained by simulation studies of two separate cases based on the UWL model; one assuming the rapid equilibrium of ligand binding with albumin, and the other considering the slow dissociation of ligands from albumin. In light of these findings, we suggest that the rate-limiting diffusion through the UWL plays an important role in the so-called albumin-mediated transport phenomenon.


Vitamin D and osteocalcin levels in liver transplant recipients. Is osteocalcin a reliable marker of bone turnover in such cases?

Patients with advanced liver disease are at increased risk for the development of hepatic osteodystrophy in the form of either osteomalacia or osteoporosis. The pathogenesis of these two bone diseases is multifactorial and includes, among other factors, alterations in vitamin D metabolism, malnutrition and hypogonadism. Little is known regarding vitamin D metabolism and the osteoblastic activity in liver transplant recipients. In order to clarify these issues, vitamin D metabolites and osteocalcin levels were measured prior to and 30 days following liver transplantation in 30 cirrhotic patients of various etiologies. While the mean plasma concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D of the entire group of 30 patients were significantly greater prior to orthotopic liver transplantation (OLTx) as compared to those after OLTx (11.5 +/- 8.6 vs. 7.4 +/- 5.8 ng/ml, p = 0.0066 and 41.0 +/- 34.6 vs. 20.4 +/- 11.0 pg/ml, p = 0.0003, respectively), no significant changes in osteocalcin concentrations pre- or post-transplantation could be demonstrated (5.2 +/- 3.0 vs. 6.4 +/- 4.1 ng/ml, p = 0.51). Furthermore, no correlation between the plasma concentration of osteocalcin and either vitamin D metabolite, the prothrombin time or cyclosporine levels was found. The reasons for the normal levels of osteocalcin prior to OLTx can be explained by the fact that in vitamin-K-deficient states osteocalcin is predominantly decarboxylated and, therefore, a smaller proportion is bound to bone and/or the synthesis of osteocalcin is partially modulated by 1,25-dihydroxyvitamin D, the level of which has been found to be normal.(ABSTRACT TRUNCATED AT 250 WORDS)


Risk of needle-stick injuries in the transmission of hepatitis C virus in hospital personnel.

To assess the risk to hospital personnel of acquiring an hepatitis C virus (HCV) infection as a result of occupational exposure to needle-stick injuries, 81 employees who had parenteral exposure to an anti-HCV-positive source were followed for 12 months. None developed hepatitis and anti-HCV testing by a second-generation ELISA system of serum samples collected on the day of exposure and at 3, 6 and 12 months was negative. Consequently, a low efficacy of needle-stick injuries in the transmission of HCV in hospital personnel may be suggested.


The validity of bioelectrical impedance analysis in estimating total body water in patients with cirrhosis.

This study assessed the validity of bioelectrical impedance analysis (BIA) to predict total body water (TBW) in patients with cirrhosis. TBW was estimated by deuterium oxide dilution (D2O) and compared with TBW predicted by BIA in 27 patients with cirrhosis with and without ascites or edema (Group A), in a subgroup of 18 'dry' cirrhotics without clinical signs of fluid overload (Group B) and in 27 healthy controls. Three different BIA regression equations were used. In all three groups of subjects high correlation coefficients were obtained between D2O-TBW and BIA-TBW (r > 0.88). In the cirrhotic Group A, BIA significantly underpredicted D2O-TBW by all 3 equations (2.9-3.8 l) and the regression lines were different from the lines of identity by two equations. Standard errors of estimate were high in Group A (3.04-3.97 l) in comparison with Group B (1.79-2.46 l) and the controls (1.03-1.41 l). In the 'dry' cirrhotics (Group B) and in the controls, TBW was correctly predicted by two of three BIA equations, and regression lines were not significantly different from lines of identity. Correlation coefficients in Group B were higher than in Group A (r = 0.96-0.97 vs. 0.89-0.92) and were comparable with controls (r = 0.98-0.99). We conclude that BIA is not a valid method of estimating TBW in cirrhotic patients with ascites and edema. In cirrhotic patients without clinical signs of fluid overload BIA can be used to predict TBW, although accuracy is slightly lower than in healthy controls.


Bacteriuria in patients with cirrhosis.

The prevalence and bacteriology of bacteriuria was studied in 140 patients with cirrhosis of the liver, referred to the University of Pittsburgh Medical Center for evaluation for liver transplantation (72 males and 68 females; mean age 46 years). Urine samples were obtained for cultures within 24 h after admission. Significant bacteriuria (SB) (> 10(5) bacteria/ml) was present in 25 patients (18%), the most common being E. coli (36%) and coagulase-negative staphylococcus (20%). SB was more common in females than in males (32 vs. 4%, p < 0.001), and was seen in every category of cirrhosis. The occurrence of bacteriuria did not correlate with the severity of the underlying liver disease or with the age of the patient. Based upon these results, it can be concluded that: (1) bacteriuria is common in patients with advanced liver disease and occurs in approximately 20% of the cases; (2) it is more common in females than in males; and (3) its prevalence in patients with primary biliary cirrhosis is similar to that in other types of chronic advanced liver disease.


Biliary endogenous inorganic phosphate, D-glucose, IgA and transferrin are differentially altered by hydrostatic pressure.

Our objective was to determine the effects of hydrostatic biliary pressure on excretion patterns of endogenous solutes which reflect various pathways of bile formation. A stable in vivo model was developed using anesthetized rats intraduodenally infused with taurocholate to maintain bile flow. Bile was collected during a 2-h basal period, a 4-h pressure period where elevation of the bile duct cannula decreased bile flow to 1/3 the basal rate, and a 2-h period after release of hydrostatic biliary pressure. During pressure treatment, bile salt concentration gradually increased approximately 3-fold, biliary inorganic phosphate concentrations rapidly rose approximately 5-fold, and biliary glucose concentration progressively rose approximately 17-fold. Concentrations of proteins in bile were affected differently with extreme decreases in IgA, moderate decreases in total protein and leucine aminopeptidase, and minimal change in transferrin. By 2 h after pressure release, only the alterations in biliary glucose and IgA persisted. The observed striking and persisting increases in biliary glucose are tentatively explained as an impaired reabsorption of glucose by the biliary tract.


Impaired functions of normal peripheral polymorphonuclear leukocytes in cirrhotic ascitic fluid.

The function of normal polymorphonuclear cells in the ascitic fluid of 32 patients with cirrhotic ascites and 17 patients with malignant ascites was studied independently of ascitic fluid heat-labile factors. Polymorphonuclear (PMN) function was assessed by a chemiluminescence method using preopsonized zymosan as stimuli. The chemiluminescence response was higher in malignant ascitic fluid than in cirrhotic ascitic fluid (0.84 and 0.15, respectively, p < 0.001). These results were confirmed by a microbiological assessment of phagocytosis. Suppressive factors were evidenced by making ascitic fluid dilutions and using cell-free chemiluminescence measurements. Addition of malignant ascitic fluid to cirrhotic ascitic fluid showed that there is also a deficiency in supportive factors other than C3. The impaired PMN production of oxidative metabolites we observed in cirrhotic ascitic fluid can partly explain the high susceptibility of cirrhotic patients to spontaneous bacterial peritonitis independently of C3 levels.