Session 4 - Pregnancy Outcome.
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Journal Article↗
Biomedical subjects
S Björnsson
Publications and source records attributed to S Björnsson.
Quantitation of proteoglycans in biological fluids using Alcian blue.
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Alcian Blue↗
Binding and detection of glycosaminoglycans immobilized on membranes treated with cationic detergents.
Immobilization of molecules on surfaces is used for preparative, quantitative, and qualitative studies. Glycosaminoglycans (GAGs) are strongly hydrophilic and negatively charged molecules that do not bind well to either polystyrene surfaces or hydrophobic blotting membranes. Hydrophobic membranes were derivatized with cationic detergents to become hydrophilic and positively charged. The ability of the polyvinylidene fluoride and nitrocellulose membranes to retain GAGs increased up to 12.8 microg per spot in the dot blot assay when the membrane was treated with a cationic detergent. Immobilized GAGs were stained with alcian blue, and the staining intensity was quantitated by scanning and densitometry. The derivatized membranes were used for solid-phase extraction of GAGs in blood plasma, urine, or cerebrospinal fluid. The detection sensitivity was equal for different types of GAGs but there was a slight negative interference from fibrinogen in blood plasma. The immobilized GAGs could also be released from the membrane using a nonionic detergent at high ionic strength. Recovery of different proteoglycan populations, separated by electrophoresis and detected by reversible staining with toluidine blue, was 70-100%.
Alcian Blue↗
Inflammatory bowel disease in Iceland, 1990-1994: a prospective, nationwide, epidemiological study.
BACKGROUND: Two separate nationwide, retrospective epidemiological studies of inflammatory bowel disease (IBD) in Iceland over a combined 40-year period, 1950-1989, have shown a continually rising incidence. The main objective of this study was to investigate the incidence of IBD prospectively for the 5-year period 1990-1994 to determine whether there still was an ongoing increase. METHODS: The retrieval of new cases of ulcerative colitis (UC) and Crohn's disease (CD) was based on a monthly review of all small and large intestinal tissue specimens with any type of inflammation submitted to all three departments of pathology in Iceland. All small intestinal X-ray records suggestive of CD were also reviewed. All possible new cases of IBD were then scrutinized by examination of the clinical records, using accepted criteria for confirmation or exclusion of IBD. RESULTS: A total of 215 cases of UC and 72 cases of CD were diagnosed, yielding a mean annual incidence for UC 16.5/100000 (95% confidence interval (CI) 14.4-18.9), and for CD 5.5/100000 (95% CI 4.3-7.0). The highest combined age related incidence for UC was 28.7/100000 in the group aged 30-39 years, and for CD 8.5/100000 in the groups aged 10-19 and 20-29 years. The most common extent in UC was proctosigmoid and the most frequent localization in CD was colonic only. CONCLUSIONS: This prospective study demonstrates a continuing and statistically significant increase in the incidence of both UC and CD in Iceland. The general changes identified for a few previously suggested environmental factors do not allow any firm conclusions as to their role in the observed increase in incidence. It is possible that there are some other causative factor(s).
Adolescent↗
Detection of epitopes in glycosaminoglycans immobilized on hydrophobic membranes.
Glycosaminoglycans (GAGs) are linear carbohydrate polymers expressed on all cell surfaces, and bind growth factors that recognize specific disaccharide sequences. Such sequences in the GAG chain are not genetically determined but may be assembled by the cell in response to environmental changes. GAGs are strongly hydrophilic and negatively charged molecules that do not bind well to either polystyrene surfaces or to hydrophobic blotting membranes. Cationic detergents were used to derivatize hydrophobic membranes to become hydrophilic and positively charged. Binding of GAGs to derivatized membranes was optimized regarding pH and ionic strength. Five different monoclonal antibodies (Mab) were used to detect sequence epitopes in immobilized GAGs. Parallel samples were stained with Alcian Blue and the staining intensities were quantitated by scanning and densitometry. By calculating the ratio between the antibody staining (epitope) and Alcian Blue staining (mass), the epitope density, i.e. the number of repetitive epitopes per mass, is obtained. The epitope density with each antibody was different with different GAGs. Some epitopes were common in GAG, i.e. highly repetitive epitopes. Some epitopes were rare and possibly expressed only once per GAG molecule, i.e. low degree of repetition. An epitope density profile was obtained when each sample was stained with all antibodies and their epitope densities calculated at the plateau level. The epitope profile is an indirect measure of the sequence variability in GAGs. Determination of epitope density and profile can be used to characterize a GAG population and to discriminate between different populations with similar chemical composition.
Animals↗
Differential expressions of mRNA for proteoglycans, collagens and transforming growth factor-beta in the human cervix during pregnancy and involution.
During pregnancy and involution, an extensive remodelling of the human cervical connective tissue occurs. This cervical ripening is one of the most pronounced physiological remodelling processes known in human connective tissue. To investigate how the remodelling is accomplished, the levels of mRNA for collagen I and III, versican and three small proteoglycans, biglycan, decorin and fibromodulin, were evaluated using Northern blots at different stages of cervical ripening. In the corresponding biopsies the concentration of collagen and of small and large proteoglycans were determined. The role of transforming growth factor-beta (TGF-beta) as a mediator of the remodelling process was also investigated. The concentration of collagen decreased and 1 week before partus, 50% of the nonpregnant level was attained. No further decrease was noted after partus. The mRNA for collagen I and III did, however, not decrease in the term pregnant cervix 1 week before partus. Only 20-30% decrease during the final ripening just before partus was recorded. Neither did the mRNA levels of the small proteoglycans change significantly during the ripening, despite an almost 50% decrease in the concentration of the small proteoglycans. The message for versican was, however, 5-fold increased at partus and then gradually returned to nonpregnant levels within 4 days after delivery. These changes corresponded to similar changes in the concentration of the large proteoglycan. Thus, the remodelling of the cervical connective tissue is achieved by two different mechanisms, on one hand an increased turnover of collagen and the small proteoglycans, on the other a changed transcription followed by an increased production of versican. During the involution 2- to 3-fold increases in the messages for collagen I and III, and the small proteoglycans, biglycan and decorin, corresponded to increases in the concentration of the small proteoglycans and non-extractable collagen. The message for TGF-beta was increased 2-fold immediately after delivery compared with the term pregnant state. Thus, TGF-beta may be of importance for the reconstruction of the cervix, which starts immediately after partus.
Adult↗
Quantitation of proteoglycans as glycosaminoglycans in biological fluids using an alcian blue dot blot analysis.
A method for quantitation of intact proteoglycans as GAGs in biological fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of tissues has been published previously (S. Björnsson, Anal. Biochem. 210, 282-291, 1993). The method is based on the specific interaction between sulfated polymers and the tetravalent cationic dye Alcian blue at pH 1.5 in 0.4 M guanidine-HCl and in the presence of 0.25% Triton. The absorbance assay has a measuring range of 1-20 microgram of glycosaminoglycan (GAG) which is not sensitive enough to measure the low contents of proteoglycans in blood plasma, urine, or wound fluid. A dot blot assay is now described in which the Alcian blue-GAG complexes are collected on a polyvinylidene fluoride membrane, by filtration in a dot blot apparatus, and the stain is quantitated as reflectance by scanning and densitometry. The assay requires 10 microliter of sample and has a measuring range of 10-800 ng of GAG, corresponding to a concentration of 1-80 mg/liter, suitable for proteoglycans in biological fluids. The procedures for chemistry, scanning, densitometry, and curve fitting were each evaluated separately. The error contributed by chemistry accounted for a minor portion of the imprecision. The imprecision contributed by scanning was the most important source of within-run and between-run imprecision, and was caused by inequalities of the charge-coupled device along the scanning arm. Unexpectedly, curve fitting was also a major source of total imprecision in dot blot quantitation and differed with the type of equation used. The between-run imprecision calculated as CV (SD/mean . 100) was 13.0% at 8 mg/liter. The response of the assay was identical for six different commercial preparations of GAGs (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin) despite differences in degree of sulfation known to exist. There was no positive or negative interference by blood plasma, apart from a slight negative interference on the quantitation of heparan sulfate. Analysis of 319 paired blood plasma and urine specimens from hospitalized patients showed a variation of plasma GAGs of 0.1-17.6 and urine-GAGs of 0.0-45.6 mg/liter. There was no correlation between plasma and urine GAG concentrations.
Alcian Blue↗
Inflammatory bowel disease in Iceland, 1980-89. A retrospective nationwide epidemiologic study.
BACKGROUND: A previous nationwide epidemiologic study of inflammatory bowel disease (IBD) in Iceland in 1950-79 showed a low but steadily rising incidence. The incidence of IBD in Iceland in 1980-89 was investigated and compared with reports from neighboring countries. METHODS: Cases were retrieved by a review of all small- and large-intestinal tissue specimens with any type of inflammation submitted to the only two departments of pathology in Iceland. All small-intestinal X-ray records suggestive of Crohn's disease (CD) in the three major hospitals were also screened. All hospital and outpatient records of cases suggestive of IBD were then reviewed using accepted criteria for confirmation or exclusion. RESULTS: The mean annual incidence of ulcerative colitis (UC) was 11.7/100,000, and that of CD 3.1/100,000. The highest age-specific incidence of UC was in the group 30-39 years old and for CD in the group 60-69 years old. The most frequent involvement at diagnosis of UC was proctitis only, in 54%, and in CD colon only, in 54.7% of the patients. CONCLUSIONS: This study shows a continuing increase in the incidence of both diseases. Compared with the period 1970-79, there has been an almost twofold increase in the mean annual incidence of UC and more than a threefold increase of CD, a statistically significant increase in both instances.
Adolescent↗
[A possible future for clinical chemistry? We must procede as a competent enterprise].
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Chemistry, Clinical↗
Decreased excretion of urine glycosaminoglycans as marker in renal amyloidosis.
BACKGROUND: The diagnosis of renal amyloidosis is normally established by kidney biopsy. In order to advance the determination of the diagnosis and the initiation of the therapy, fast and cheap, non-invasive diagnostic techniques are required. METHODS: Urine excretion of glycosaminoglycans (GAG) was measured in 10 patients with AA amyloidosis and 5 patients with AL amyloidosis and compared to 25 controls with primary glomerular diseases and 22 healthy controls. The subjects with primary glomerular disease were matched with regard to their renal function and the degree of albuminuria. RESULTS: The median urine GAG to creatinine ratio and the median fractional GAG excretion were significantly decreased (P < 0.05) in both AA amyloidosis (0.21 mg/mmol and 0.053 respectively) and AL amyloidosis (0.33 mg/mmol and 0.077 respectively) compared to control patients with primary glomerular disease (1.73 mg/mmol and 0.336 respectively) and healthy controls (2.67 mg/mmol and 0.226 respectively). The urine GAG to creatinine ratio did not correlate to age, sex, serum creatinine, urine albumin, or to the plasma levels of acute phase proteins. CONCLUSIONS: The decreased GAG excretion in renal amyloidosis is probably caused both by diminished number of functioning nephrons, decreased GAG synthesis in functioning glomeruli, and the trapping of GAG by amyloid fibrils. Urinary GAG excretion may serve as an independent marker of renal amyloidosis. It may be used in diagnostic work-up of renal amyloidosis in patients with glomerular diseases and in screening of amyloidosis in patients with chronic inflammatory disorders, with or without signs of renal disease.
Adult↗
Dietary fish oil supplementation increases survival in mice following Klebsiella pneumoniae infection.
The effect of dietary fish-oil supplementation on survival of NMRI mice after Klebsiella pneumoniae infection was investigated. 30 mice in each group were fed a fish-oil enriched diet, olive-oil enriched diet or standard chow diet. After 6 weeks, the mice were injected intramuscularly with Klebsiella pneumoniae. After 120 h the survival of the mice fed fish-oil enriched diet was 40%, while the survival for mice fed standard or olive-oil enriched diets was 20% and 25%, respectively. The survival curve over 120 h was significantly improved (p = 0.0034) for mice fed a fish-oil enriched diet, compared to the survival curves for mice fed the other 2 diets. The study was repeated by comparing the survival of mice fed a fish-oil enriched diet to those given a corn-oil enriched diet. After 120 h the survival curve for mice fed the fish-oil enriched diet was significantly better compared to the survival curve for mice given the corn-oil enriched diet (p = 0.01). A fish-oil enriched diet therefore increases survival in mice following Klebsiella pneumoniae infection, whether compared to a standard diet, olive-oil enriched diet or corn-oil enriched diet.
Animals↗
Decreased excretion of glycosaminoglycans in patients with primary glomerular diseases.
Urine glycosaminoglycans (GAG) concentrations were measured in 150 patients with primary glomerulonephritides: endocapillary glomerulonephritis, mesangial proliferative glomerulonephritis, IgA nephropathy, membranous glomerulonephritis and minimal change nephropathy, and in 63 healthy controls and 19 patients with diabetes nephropathy. The urine GAG to creatinine ratios (GCR) were significantly reduced (p < 0.01) in all the glomerulonephritides investigated (0.20 mg/mmol in endocapillary glomerulonephritis, 1.60 mg/mmol in mesangial proliferative glomerulonephritis, 1.74 mg/mmol in IgA nephropathy, 1.09 mg/mmol in membranous nephropathy, and 1.16 mg/mmol in minimal change nephropathy) compared to healthy controls (2.87 mg/mmol) but not compared to diabetes patients (1.17 mg/mmol). Also, the GCR in a group of 23 non-albuminuric glomerulonephritis patients (1.98 mg/mmol) was shown to be significantly decreased (p < 0.01) compared to healthy controls. Moreover, the GCR was significantly lower (p < 0.01) in endocapillary glomerulonephritis than in any of the other diseases studied. The GAG excretion per functioning glomerular area, calculated as fractional GAG excretion (FGE), was decreased in all the glomerulonephritides investigated compared to both healthy controls and diabetes nephropathy. The decreased GAG excretion in glomerulonephritides, obtained in the present study, might be a consequence of decreased synthesis or turnover of GAG in the functioning nephrons whereas the mechanisms for the reduced GAG excretion in diabetes nephropathy might be of a different nature. Urinary GAG excretion in this group of glomerular disorders and particularly in endocapillary glomerulonephritis, may lead to new approaches in non-invasive renal diagnostics and, particularly with regard to the differentiation of acute and chronic forms of glomerulonephritides.
Adolescent↗
Systematic screening for diabetic eye disease in insulin dependent diabetes.
Under a national program established in 1980, the eyes of approximately 90% of the insulin dependent diabetic patients in Iceland have undergone annual eye examination and fundus photography. Laser treatment was given for proliferative retinopathy or diabetic macular edema according to Diabetic Retinopathy Study and Early Treatment Diabetic Retinopathy Study criteria. We report on 205 insulin-taking patients whose age at diagnosis was less than 30 years of age. Retinopathy was present in 106 (52%) patients and proliferative retinopathy in 26 (13%). 196 patients (96%) had visual acuity equal or better than 6/12 in their better eye, 6 patients (3%) with 6/18-6/48 in their better eye, and 2 patients (1%) equal or worse than 6/60 in their better eye.
Adolescent↗
Screening for eye disease in type 2 diabetes mellitus.
A screening program for diabetic eye disease was established in Iceland in 1980. Approximately 90% of the insulin dependent patients in Iceland undergo annual eye examination and fundus photography and about a fifth of the type 2 diabetic patients. We report on 245 diabetic patients with type 2 diabetes. Any diabetic retinopathy was present in 100 patients (41%), proliferative retinopathy had been present in 17 (7%) and 24 (10%) had clinically significant diabetic macular edema. Two-hundred and twenty-four patients (91%) had visual acuity equal or better than 6/12 in their better eye, 17 patients (7%) with 6/18-6/36 in their better eye, and 4 patients (1.6%) equal or worse than 6/60 in their better eye.
Adolescent↗
Renin gene restriction fragment length polymorphisms do not show linkage with preeclampsia and eclampsia.
OBJECTIVE: To investigate linkage between the renin gene restriction fragment length polymorphisms in families with a history of preeclampsia/eclampsia. METHODS: Nine Icelandic families with at least three affected females in two or three generations were investigated. DNA from lymphocytes was digested with the endonuclease restriction enzyme Bgl I and restriction fragments were transferred by Southern Blotting. Hybridisation was effected with the 32P-oligonucleotide-labeled diallelic genomic probe pHRnX 0.8. LOD scores were calculated by the Liped program for two forms of inheritance patterns. Affected sib pairs were analysed. RESULTS: Frequencies of the 9.0 kb and 5.0 kb alleles were 0.67 and 0.33, with no significant differences between affected females and spouses and combined LOD scores of -2 for recombination values of 3%. Allele sharing in affected sibs was not different from the expected random assortment. CONCLUSION: The linkage analysis provides evidence to exclude alteration of the renin gene in pregnancy as being directly responsible for the manifestations of preeclampsia or eclampsia in these families.
Alleles↗
Metabolic turnover of sulfated glycosaminoglycans and proteoglycans in rabbit temporomandibular joint cartilages with experimentally induced osteoarthrosis.
Osteoarthrosis-like changes were induced by means of experimental disk perforation in the right temporomandibular joint of rabbits. The turnover of proteoglycans and glycosaminoglycans was studied 16 weeks later, using 35SO4. Tissues were sampled 1 day and 7 days after injection of the sulfate. The corresponding tissues from the left untreated joint were used as controls. After isolation of the glycosaminoglycans the incorporation of 35SO4 was estimated by scintillating counting. The extracted proteoglycans were analyzed, using gel electrophoresis, and the distribution of radioactivity was determined by autoradiography, followed by densitometry. Both the synthesis and rate of degradation of the proteoglycans were increased in the experimental disk, compared with those of the control. The net result of these metabolic changes seemed to be losses of small proteoglycans, whereas a slow increase in the number of larger ones may have occurred. The turnover rates of 4- and 6-sulfate increased, although their ratio remained unchanged at this stage of the osteoarthrosis-like process. In the condylar cartilage the turnover of large and small proteoglycans was also increased. The increase was most marked among those containing 6-sulfated galactosaminoglycans. The results concerning the experimental condylar cartilage indicated a decrease in the largest proteoglycan population, whereas the proportion of small proteoglycans was increased.
Animals↗
Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue.
Conditions for specific interaction between Alcian blue and proteoglycans were optimized by comparing the differential spectra of Alcian blue obtained with purified chondroitin sulfate dissolved in water with the spectra obtained with nasal cartilage proteoglycans dissolved in synovial fluid. A method was then designed that provides specific precipitation of proteoglycans or glycosaminoglycans in 4 M guanidine - HCl in the presence of protein, hyaluronic acid, or nucleic acids. The specificity is achieved by using a low pH in combination with detergent and high salt concentration. Stepwise addition of reagents is necessary to avoid binding of Alcian blue to proteins and nucleic acids. All polyanions, except polysulfates, are first neutralized by lowering the pH to 1.5. By including detergent in this step, the hydrophobic protein regions are blocked and not accessible for binding with the dye. These regions could otherwise bind Alcian blue by hydrophobic interaction. When the Alcian blue reagent is added after, only the polysulfated molecules will remain charged and free to interact with Alcian blue. At least 0.4 M guanidine-HCl is required to abolish the negative interference by proteins. All sulfated glycosaminoglycans are precipitated at 0.4 M guanidine-HCl. With increasing guanidine-HCl concentrations, the different glycosaminoglycans are precipitated in accordance with the critical electrolyte concentration of the respective glycosaminoglycan. The Alcian blue precipitation can be performed at different concentrations of guanidine-HCl in order to separate different classes of proteoglycans. Excess dye and contaminating proteins are removed by a wash in a DMSO-MgCl2 solution and the precipitate is dissolved in a mixture of guanidine-HCl and propanol. For quantitation, the absorbance is recorded in a microplate reader with the 600-nm filter, the assay being linear between 0.5 and 20 micrograms proteoglycan. Since no digestion of samples with protease is needed, the proteoglycans are recovered in native form. The proteoglycan-Alcian blue complexes dissociate in the guanidine-HCl/propanol mixture and the proteoglycans can be selectively precipitated with propanol. The dye is used for quantitation and the proteoglycans can be utilized for further analysis.
Alcian Blue↗
Size-dependent separation of proteoglycans by electrophoresis in gels of pure agarose.
An improved system for electrophoresis of proteoglycans is presented, using a discontinuous buffer system that allows stacking of the sample. The molecular sieving in pure agarose was studied using cartilage proteoglycans separated into fractions of different size (Kav value) by gel chromatography. These fractions were used to establish the relationship between size and mobility in agarose gels of different concentrations. With low agarose concentration, the separation was largely dependent on charge density. With increasing agarose concentration, the separation became increasingly dependent on size. Electrophoresis provides a suitably sensitive tool for analysis of the proteoglycan and glycosaminoglycan fractions prepared by the Alcian blue precipitation described in the preceding paper. Alcian blue precipitated cartilage proteoglycans appeared identical in size to molecules prepared by conventional procedures when analyzed by electrophoresis. Proteoglycans of various sizes were prepared by precipitation with Alcian blue of 4 M guanidine-HCl extracts of bovine nasal cartilage, human articular cartilage, human skin, and bovine sclera. Proteoglycans were also precipitated from human synovial fluid and plasma. The samples were electrophoresed on 1.2% agarose gels, which was the highest agarose concentration suitable for large aggregating proteoglycans but still separating the smallest proteoglycans with one or two glycosaminoglycan chains from each other. Several sizes of proteoglycans were found in each sample except blood plasma, which only contained one small proteoglycan/glycosaminoglycan. Electrophoresis in agarose is also suitable for separating proteoglycan aggregates and monomers and allows rapid analysis of several samples at a time. The proteoglycans prepared by Alcian blue precipitation retained their ability to form aggregates with hyaluronic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcian Blue↗