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Biomedical subjects

G Eberlein

Publications and source records attributed to G Eberlein.

17 recordsLinked to original sources
PubMed2000

Characterization of bovine collagens using capillary electrophoresis.

A capillary electrophoresis method was developed and characterized for analyzing the spectrum of collagen subspecies in collagen preparations. The Bio-Rad CE-SDS protein kit was used for the dynamic sieving separation of collagen subspecies in this CE method (DSCE). The optimized method utilized a 36 cm (or 24 cm) x 50 microm uncoated capillary, electrophoretic injection at 10 kV for 10 s, a run voltage of 15 kV, a capillary temperature of 20 degrees C, and UV detection at 220 nm. A preliminary validation of the method was performed. The assay had good repeatability (RSDs for peaks were 15%), and responses were linear for assay solutions with collagen concentrations from 0.125 to 1.25 mg/ml. The DSCE electropherogram of bovine skin collagen provided a profile of subspecies similar in number and relative abundance to that generated by scanning of Coomassie-stained SDS-PAGE gels.

Animals↗
PubMed1999

Use of acarbose for eliminating the interval between meal consumption and insulin injection in patients with Type 1 diabetes.

This double-blind, placebo-controlled, cross-over study investigated whether acarbose therapy enables patients with Type 1 diabetes to administer insulin injections at the same time as meals without adverse glycaemic consequences, thus negating the need for an injection-meal interval (IMI). The valid-case population was 15. All patients were well controlled on insulin therapy and diet. Each patient underwent four 14-d treatment sequences: IMI of 0 min+acarbose; IMI of 30 min + acarbose; IMI of 0 min + placebo; IMI of 30 min + placebo. There was a 1-wk, placebo-controlled washout period between each treatment. A 50 mg acarbose or placebo tablet was taken with each of three meals for the first 3 d of each treatment; this was increased to 100 mg three times daily on the remaining 11 d. Blood samples were taken between 08.00 and 09.00 h at the end of each treatment sequence, during the fasting state and at the following times after breakfast: 30, 60, 120 and 180 min. The primary target variable was 1-hr postprandial blood glucose level after 14 d. The mean increase in 1-hr postprandial blood glucose level using an IMI of 0 min + acarbose was 51.1+/-53.2 mg/dl, compared with 46.6+/-50.4 mg/dl using a 30-min IMI + placebo. Data provided strong evidence that acarbose prevents the marked increase in postprandial glucose level normally observed when insulin is administered with a meal. Acarbose may thus be useful for patients with Type 1 diabetes who find IMIs inconvenient.

Acarbose↗
PubMed1994

Probing the conformation of protein (bFGF) precipitates by fluorescence spectroscopy.

Aggregation and precipitation are major events in the handling and aging of most protein pharmaceuticals. We demonstrate the utility of fluorescence spectroscopy in determining protein conformation in precipitates using basic fibroblast growth factor (bFGF) as an example. Conversion of the native to the soluble denatured from by chaotropes was accompanied by an increase in tryptophan emission. The emission spectra of resuspended precipitates were as reproducible as the spectra of the soluble form. The sum of emission spectra of native soluble bFGF and denatured precipitated bFGF was superimposable on the spectrum of the unfractionated suspension, suggesting that quantitative analysis of denatured aggregates in turbid protein formulations is possible. The ratio of tryptophan to tyrosine emissions increased with increasing extent of denaturation both in solution and in suspension. For example, salting out by ammonium sulphate increased the fluorescence index (indicative of denaturation) which was reversible upon dissolution. In addition, aging (35 degrees C) of bFGF in the presence of sulphated ligands produced precipitates with native-like fluorescence index, in contrast to denatured precipitates formed without ligands.

Chromatography, High Pressure Liquid↗
PubMed1994

Chromatographic methods for quantitative analysis of native, denatured, and aggregated basic fibroblast growth factor in solution formulations.

High-performance liquid chromatography (HPLC) methods were developed for evaluating stability of human recombinant basic fibroblast growth factor (bFGF) against denaturation and aggregation in solution formulations. Reversed-phase chromatography (RP-HPLC)-insensitive to bFGF tertiary structure--was used to measure total soluble protein; heparin affinity chromatography (HepTSK) provided quantitative analysis of native bFGF species. The folding state of bFGF was determined by fluorescence spectroscopy: Tryptophan emission, which was quenched in native protein, increased upon unfolding. Slow unfolding/refolding kinetics of bFGF in 2 M guanidine hydrochloride made possible the separation of native from denatured species by size exclusion chromatography (SEC). Although the tertiary structure affected bFGF retention times, it did not change the sample recovery by SEC. These chromatographic techniques, which quantitatively measure physical and chemical changes taking place in solution formulations, can be used in future investigations of bFGF stability.

Chemical Phenomena↗
PubMed1994

Major degradation products of basic fibroblast growth factor: detection of succinimide and iso-aspartate in place of aspartate.

The degradation products of basic fibroblast growth factor (bFGF) were isolated by ion exchange HPLC (HP-IEC) and characterized. The predominant product at pH 5 was a succinimide in place of aspartate15 as determined by LC/MS, N-terminal sequencing, and susceptibility to degradation at pH > 6.5. The rate of appearance of the succinimidyl-bFGF at 22 degrees C was comparable to that reported for small peptides, consistent with a high flexibility predicted for asp15-gly. Tryptic mapping together with [3H]-methylation indicated that iso-aspartate was formed at the position of asp15. Size exclusion HPLC indicated the presence of intact and truncated dimers and trimers which associated through disulfide linkages. Two truncated monomer forms were found that co-eluted by HP-IEC; the cleavages were determined to be at asp28-pro and asp15-gly using LC/MS and N-terminal sequencing. These degradation products which occurred at sites that are away from receptor or heparin binding domains of bFGF remained bioactive in a cell proliferation assay.

Amino Acid Sequence↗
PubMed1994

The stability of bFGF against thermal denaturation.

The influence of sulphated ligand and pH on thermal denaturation of basic fibroblast growth factor (bFGF) was investigated by differential scanning calorimetry (DSC), and verified by fluorescence spectrophotometry. Purity of bFGF before and after heat denaturation was assessed by SDS-PAGE analysis. In DSC studies the samples were heated to 95 degrees C. The midpoint of the temperature change in the thermogram was designated as Tm. Sulphated ligand experiments were undertaken in potassium phosphate (pH 6.5) and sodium acetate buffers. Control thermograms (with no ligand) showed a Tm at 59 degrees C in potassium phosphate buffer. Higher Tm values were noted as sulphated ligand concentration was increased. Similarly when heparin was added, the Tm moved to a higher temperature. A ratio as low as 0.3:1 of heparin to bFGF, increased the Tm to 90 degrees C, which is a 31 degrees C shift in Tm. The effect of pH on thermal denaturation of bFGF was studied in a citrate-phosphate-borate buffer system. A shift in Tm from 46 to 65 degrees C was observed as the pH is changed from 4 to 8. Changes in protein conformation as a function of pH were monitored by fluorescence spectroscopy. It was found that a pH range from 5 to 9 is optimal for the stability of bFGF formulations. In a stability study it was noted that heparin protected bFGF from thermal denaturation only at high temperature.

Buffers↗
PubMed1991

Determination of various molecular forms of cholecystokinin from canine mucosa by radioimmunoassay and bioassay.

Bioassays using amylase release from isolated pancreatic acini measure only cholecystokinin (CCK) forms with an intact carboxyl terminus ending with phenylalanine amide, but it cannot be excluded that peptides not structurally related to CCK are also responsible for CCK-like bioactivity. CCK exists in several molecular forms in intestinal mucosa which are released into the circulating blood. We studied the molecular forms of CCK in canine intestinal extracts after separation by high performance liquid chromatography by bioassay and compared them with those detected by radioimmunoassay. For the radioimmunoassay, an antibody was used which needs the carboxyl terminal phenylalanine amide for recognition. Three immunoreactive peaks were reproducibly seen in HPLC eluates which eluted in the regions of synthetic CCK-8, purified porcine CCK-33-39 (which co-elute using this gradient) and purified canine CCK-58. All these peaks were bioactive for amylase release from isolated pancreatic acini. No further bioactive peaks were detected in the HPLC eluates. When an antibody was used which recognizes the midregion of CCK-58, an additional peak was detected which eluted between CCK-33-39 and CCK-58. This form presumably represents an amino terminal fragment of CCK lacking the carboxyl terminus. It can be concluded that bioassays of CCK measure only CCK bioactivity in intestinal mucosal extracts, whereas radioimmunoassays may detect biologically inactive forms depending on the antibody recognition site.

Amylases↗
PubMed1991

Inverse relationship between diastolic blood pressure and urinary excretion of potassium in girls aged 8 to 9 years--a preliminary communication.

In order to investigate the association of urinary excretion of sodium and potassium with blood pressure, 72 prepubertal healthy children between 8 and 9 years were investigated in a cross sectional study. We determined anthropometric data, diastolic (DBP), and systolic blood pressure (SBP) and urinary excretion of sodium and potassium in 24-hour urine samples. No relationship was found between urinary sodium or potassium excretion and systolic or diastolic blood pressure, neither with regard to simple, nor multiple regression analysis. However, on simple linear and multiple regression analysis a significant inverse association between the excretion of potassium and diastolic blood pressure (r = -0.53; p less than 0.01 and r = -0.60; p less than 0.001) was observed in girls. From our data we conclude that in normal weight, healthy children urinary excretions of sodium and potassium are not associated with systolic blood pressure. However, a high 24-hour urinary excretion of potassium might be associated with lower diastolic blood pressure in prepubertal girls.

Anthropometry↗
PubMed1990

[Cholesterol, triglycerides and lipoproteins in the serum of healthy, normal weight children between 6 and 12 years of age].

Serum cholesterol, triglycerides and lipoproteins were investigated in 276 healthy, prepubertal children and adolescents of normal weight aged 6 to 12 years. The results were evaluated on the basis of the guidelines of the Austrian Cholesterol Consensus Conference 1988 and according to the 90th and 95th percentiles of the Lipid Research Clinics (LRC) data. A relatively high percentage of the probands revealed cholesterol concentrations which could be classified as "risk" (50% of the female and 53% male probands) or "high risk" (39% of the girls and 25% of the boys). Classifying the cholesterol data according to the LRC criteria 46% of the girls and 41% of the boys exceeded these "cut off" values of the 90th percentile. Our data show that the mean serum cholesterol concentrations in healthy children and adolescents of normal weight--like in other European countries--are in a region concomitant with the category in adults which is associated with an increased risk for cardiovascular diseases. We assume that apart from genetic factors a high fat intake might play a predominant role in causing these raised levels.

Austria↗
PubMed1990

[Paralytic ileus as an initial manifestation of malignant VIPoma of the pancreas--case report with review of the literature].

A 57-year old patient with a paralytic ileus of unknown origin was admitted to the intensive care unit. Because of the laboratory findings with therapy resistant hypokalemia, hypercalcemia and metabolic acidosis a VIPoma was suspected. Therapy with somatostatin resulted in correction of laboratory abnormalities and in normalization of gastrointestinal motility. Plasma concentrations of VIP and PP were elevated, ultrasonography revealed a pancreatic tumor. Postsurgical examination of the removal tumor tissue confirmed the diagnosis of a malignant VIPoma. Clinical symptoms, laboratory findings with and without somatostatin-therapy and immunhistochemical properties are described.

Biomarkers, Tumor↗
PubMed1989

Influence of food on plasma cholecystokinin and gastrin in patients with partial gastric resections and Roux-en-Y anastomosis.

Cholecystokinin (CCK) is a polypeptid released postprandially by the upper intestinal mucosa. There are several biological active forms of CCK. Radioimmunological measurements of CCK may not detect all biological active forms or may have the disadvantage of crossreacting with gastrin. In the following we describe a modification of a bioassay for CCK which was first developed by Liddle et al. (J Clin Invest 1985). By means of this bioassay pre- and postprandial plasma CCK-levels of healthy male volunteers are compared with CCK-levels of patients with partial gastric resections and excluded duodenum. Both groups showed similar basal CCK-values (about 1 pM) and a food induced increase of this hormone by reaching maximal values after 15 to 30 min (control: 4.30 +/- 0.65 vs. operated: 13.37 +/- 2.83 pM). Patients with gastric resections, however, had about three times more CCK released over the 60 min time period studied as compared to controls. Thus exclusion of the duodenum, the supposed main place of CCK production, does not cause a lower but rather higher increase of postprandial CCK release.

Adult↗
PubMed1988

An amino-terminal fragment of cholecystokinin-58 is present in the gut: evidence for a similar processing site of procholecystokinin in canine gut and brain.

Radioimmunoassays using antibodies specific for the carboxyl terminus of cholecystokinin (CCK) and the midportion of CCK-58 (raised against synthetic canine CCK-33-(1-27] revealed the existence of a CCK fragment in canine gut and brain extracts which lacks the biologically active carboxyl terminal immunoreactivity. This material eluted on Sephadex G-50 gel permeation chromatography in the region of CCK-58, on high-pressure liquid chromatography (HPLC) after CCK-39 and before CCK-58, and on cation-exchange FPLC it eluted after CCK-58. The immunoreactive pattern, the ratio of absorbance at 280-220 nm and the chromatographic elution positions suggest that this large CCK-like molecule represents an amino-terminal fragment of CCK-58. This fragment is present in canine gut and brain. Therefore, a similar processing site of procholecystokinin is suggested in both tissues.

Animals↗
PubMed1986

Cholecystokinin--gene structure, and molecular forms in tissue and blood.

The gene structure of human cholecystokinin (CCK) is known and the cDNA structures are known for pig and rat. However, the molecular forms of CCK produced, stored and released from neural or endocrine cells cannot be predicted from the known structures of preprocholecystokinin derived from cDNA. It is well recognized that gastrin and CCK share carboxyl-terminal homology and similar processing to form the biologic active peptides. Comparisons of the structures of preprogastrin and preprocholecystokinin reveal a similar degree of homology in the midregion of the precursors (the structure of preprogastrin 55-59 and preprocholecystokinin 39-42 is His-Arg-Arg-Gln-Leu). Gastrin-34 (whose amino terminus corresponds to position 58 of preprogastrin) is formed by cyclization of glutamine to pyrrolidone carboxylic acid subsequent to cleavage at the double basis site. Identical posttranslational processing in this region would yield CCK-61. However, CCK-58 is the largest biologically active peptide so far isolated and chemically characterized. CCK can be found in high concentrations in gut and brain but the molecular forms differ in the two tissues. Thus, the most abundant forms in the intestine are CCK-58, followed by CCK-39, CCK-33, and CCK-8, whereas in the brain CCK-8 is the most abundant form, followed by CCK-58 and CCK-5. The brain contains in contrast to the intestine little or no CCK-39 and CCK-33. Therefore differences in posttranslational processing between gut and brain are assumed. The predominant small CCK forms in the brain act presumably as neurotransmitters. Large and small molecular forms are released from the endocrine cell of the gut and act as circulating hormones. Several problems have hindered the characterization and quantitation of CCK forms in tissue and blood. The large forms (CCK-58, CCK-39, CCK-33) are easily lost because they are more basic and bind to glass and plastic surfaces. In addition to recovery problems, CCK-58 can be readily degraded into smaller forms at neutral or basic pH in tissue extracts and in blood or plasma. When special care is taken to prevent these problems, we have shown that, in the dog, CCK-58 is not only the most abundant molecular form in the intestinal mucosa but also in the circulating blood. The physiology and pathophysiology of circulating CCK (regulation of pancreatic secretion, of the motility of the gallbladder and the bowel, and involvement in satiety mechanisms) has to be studied further.

Amino Acid Sequence↗