Specific Radioassay Research Articles

ICA512(IA-2) Epitope Specific Assays Distinguish Transient from Diabetes Associated Autoantibodies

ICA512/IA-2, a tyrosine phosphatase-like protein, is one of the major autoantigens in type 1 diabetes. Following phage display characterization of ICA512 autoantigenic epitopes, we developed fluid phase autoantibody radioimmunoassays for a series of ICA512 fragments (F1 [amino acids (aa): 761–964], F2A [aa 256–760], F2B [aa 761–928], and F2C [aa 929–979]). With the hypothesis that ‘non-diabetes associated’ ICA512 autoantibodies would differ from diabetes associated ICA512 autoantibodies in terms of epitopes recognized, we analyzed ten such serum samples (two from normal control individuals, one from a general population subject with transient ICA512 autoantibodies and seven from relatives of patients with type 1 diabetes who had single transient ICA512 positivity). All but one of the ‘non-diabetes associated’ ICA512 positive samples (9/10) did not react with Fragment 1 which contains the major antigenic epitopes of the molecule that were recognized by almost all (51/52) ICA512 positive new onset patient samples and pre-diabetic relatives (P< 10−6). The great majority of samples (44/52) from the new onset patients and pre-diabetic relatives reacted with at least two fragments and 60% (31/52) with three or more fragments. In contrast, only one sample of the ICA512 ‘non-diabetes associated’ sera reacted with multiple fragments (P< 10−4). Our findings suggest that diabetes associated anti-ICA512 autoantibodies react with multiple ICA512 epitopes while non-diabetes associated ICA512 autoantibodies may usually represent reactivity of antibodies with determinants of ICA512 unrelated to type 1 diabetes. The ability to distinguish diabetes associated from non-diabetes associated anti-ICA512 autoantibodies should provide prognostic information and more importantly suggests that even with highly specific radioassays positivity may occur unrelated to type 1 diabetes.

Biochemical evidence for two novel enzymes in the biosynthesis of 3-dimethylsulfoniopropionate in Spartina alterniflora.

3-Dimethylsulfoniopropionate (DMSP) is an osmoprotectant accumulated by the cordgrass Spartina alterniflora and other salt-tolerant plants. Previous in vivo isotope tracer and metabolic modeling studies demonstrated that S. alterniflora synthesizes DMSP via the route S-methyl-Met --> 3-dimethylsulfoniopropylamine (DMSP-amine) --> 3-dimethylsulfoniopropionaldehyde --> DMSP and indicated that the first reaction requires a far higher substrate concentration than the second to attain one-half-maximal rate. As neither of these reactions is known from other organisms, two novel enzymes are predicted. Two corresponding activities were identified in S. alterniflora leaf extracts using specific radioassays. The first, S-methyl-Met decarboxylase (SDC), strongly prefers the L-enantiomer of S-methyl-Met, is pyridoxal 5'-phosphate-dependent, generates equimolar amounts of CO(2) and DMSP-amine, and has a high apparent K(m) (approximately 18 mM) for its substrate. The second enzyme, DMSP-amine oxidase (DOX), requires O(2) for activity, shows an apparent K(m) for DMSP-amine of 1.8 mM, and is not accompanied by DMSP-amine dehydrogenase or transaminase activity. Very little SDC or DOX activity was found in grasses lacking DMSP. These data indicate that SDC and DOX are the predicted novel enzymes of DMSP synthesis.

Serum levels of insulin-like growth factor binding protein-3 in benign and malignant breast disease.

The insulin-like growth factors IGF-I and IGF-II and their major binding protein IGFBP-3 influence the growth of breast cancer cells in vitro. Some benign non-breast tumours appear to be associated with increased serum IGFBP-3 levels which would tend to reduce bioactive-free IGF concentrations. The present study investigates whether this pattern also occurs in neoplastic breast disease. Serum IGF-I, IGF-II and IGFBP-3 were measured by specific radioassay in 12 women with benign breast disease, 31 patients with breast cancer and in age-matched controls. The mean (+/-SD) serum IGFBP-3 concentration was higher in benign breast disease (3.6+/-0.7 mg/L) than in controls (2.7+/-0.6 mg/L) or in breast cancer patients (2.7+/-0.5 mg/L) (P = 0.001). Serum IGF-I and IGF-II levels were not significantly different among the groups. However, the index of free unbound IGF measured as the molar ratio of IGF-I plus IGF-II divided by IGFBP-3 was significantly lower in benign breast disease than in the other subjects. Either the production or clearance of IGFBP-3 is altered in benign breast disease so that there is less free IGF available to cells. This may serve to protect against malignant transformation in patients with benign breast disorders.

Direct fingerprinting of metabolically active bacteria in environmental samples by substrate specific radiolabelling and lipid analysis

Substrate specific radio assays were used for enumeration and fingerprinting of microorganisms in environmental samples. Direct fingerprinting was based on incorporation of 14C-labelled substrates into microbial lipids. A radioactive fingerprint was obtained by subsequent radio analysis of whole sample phospholipid ester-linked fatty acids ( 14C-PLFA fingerprint). This approach provided a 14C-PLFA fingerprint of the organism actively metabolizing the added 14C-labelled substrate. Labelled and unlabelled PLFAs were analysed as methyl ester derivatives by gas–liquid chromatography with flame ionization detection. The presence of 14C-PLFAs were determined by collection of 14CO 2 produced after combustion of the fatty acids. Additional analysis of the microbial community was carried out by analysis of the radioactivity assimilated into poly-β-hydroxyalkanoates relative to that assimilated into total phospholipids ( 14C-PHA/ 14C-PL ratio). The number of organisms involved in the degradation of a 14C-labelled substrate was estimated using a 14C-most-probable-number technique. These different 14C-based methods were evaluated by studying [ 14C]methane oxidation in agricultural soil, and [ 14C]phenanthrene degradation in activated sludge and marine sediment. The radio assays resulted in distinct fingerprints of the bacterial populations capable of degrading the different radiolabelled substrates. Manipulation of the incubation conditions (e.g., oxygen status) resulted in significant changes in population specific metabolic activity and labelling pattern. Phenotypically related microorganisms appeared to dominate [ 14C]phenanthrene degradation in activated sludge and marine sediment under oxic conditions. Anaerobic [ 14C]phenanthrene degraders in activated sludge produced a very different 14C-PLFA fingerprint. In methane enriched agricultural soil, aerobic methane oxidation was dominated by organisms most similar to the Type I methanotrophic bacteria. Several of the findings obtained by the 14C-PLFA analysis could not have been established on the basis of conventional PLFAs analysis alone. The results suggest that variations of this simple 14C-fingerprinting method may be applicable to studies of substrate metabolism in mixed microbial communities. Direct fingerprinting based on substrate specific radiolabelling may also aid in phenotypic characterization of heterotrophic microorganisms without the need for enrichment or cultivation.

Circulating telopeptide type I is a peripheral marker of thyroid hormone action in hyperthyroidism and during levothyroxine suppressive therapy.

The aim of the present investigation was to evaluate the clinical performance of serum carboxy-terminal-1-telopeptide (ICTP), a new marker of bone resorption, in identifying peripheral overexposure to thyroid hormones, as compared with serum osteocalcin (OC) and serum sex hormone binding globulin (SHBG). Serum ICTP, SHBG, and OC were assayed by specific radioassays in three study groups. Group 1: 50 perimenopausal women on long-term levothyroxine (LT4) suppressive treatment; group 2: 29 women with untreated hyperthyroidism; group 3: 36 normal euthyroid women matched with group 1 patients for age, alcohol, smoking habits, and lifestyle. Serum concentrations of SHBG, ICTP, and OC were markedly increased in hyperthyroid patients, whereas only serum ICTP was slightly but significantly increased in LT4 treated patients. Serum ICTP had higher diagnostic value for hyperthyroidism when compared with SHBG and to OC (sensitivity: 100%, 71%, 55%; accuracy: 97%, 88%, and 76%, respectively). In group 1, increased serum ICTP was observed in 30 of 50 patients, whereas increased SHBG and OC were found only in 11 of 50 (p < .001). Serum free thyroid hormone concentrations correlated with circulating ICTP and SHBG, and the correlation with serum OC was of lower significance. In conclusion, serum ICTP is a sensitive and reliable marker of peripheral thyroid hormone activity at the bone level; its clinical performance is higher than OC and even better than SHBG. Thus, serum ICTP is better than other peripheral markers in monitoring LT4 suppressive therapy in patients at increased risk for osteoporosis such as perimenopausal women.

Vitamin B-12 content and bioavailability of spirulina and nori in rats

The availability of vitamin B-12 from two different seafood products, nori ( Porphyra tenera) and spirulina ( Spirulina sp.), was evaluated in rats. Male weanling Wistar rats ( n = 30) were fed a vitamin B-12 deficient diet for six weeks, followed by a four-week repletion period in which the rats were supplemented with equal doses of vitamin B-12, either supplied as pure cyanocobalamin, or as dried spirulina or nori. No difference in body weight gain, relative liver, or relative kidney weight could be demonstrated between the groups in either the depletion or repletion period. Low serum, liver, and kidney cobalamin contents were measured after the depletion period using an Intrinsic Factor-based radioassay. However, no hematological abnormalities could be demonstrated in the B-12 depleted rats. After repletion, cobalamin contents of serum and kidney were significantly lower, and liver cobalamin content was higher, for both the nori- and spirulina-fed rats than for the cyanocobalamin-supplemented controls. These data illustrate that cobalamins from algae are indeed absorbed by the rat. However, the distribution pattern over liver and kidneys indicates that at least part of the cobalamins, measured by a specific radioassay, may actually be analogues.

Determination of concentration of hyaluronate in equine serum

Summary Concentration of hyaluronate (ha) in equine serum was determined by a recently developed specific radioassay. The mean ± SD HA concentration in equine serum was 288 ± 145 μg/L, was age dependent, and varied widely between horses (range, 190 to 760 μg/L). Light or moderate exercise increased serum ha concentration from baseline values by 1.5- to 3-fold. In all horses, serum ha concentration returned to or below the original resting values 1 and 2 hours after exercise.

Hyaluronan in experimental serous and purulent otitis media.

The concentration of hyaluronan (hyaluronic acid; sodium hyaluronate; HA), a major component of connective tissue, was analyzed by a specific radioassay in tissue samples from normal rat middle ears and in tissue and effusions from middle ears affected by experimentally induced otitis media. In normal ears the pars flaccida of the tympanic membrane contained a considerably higher concentration of HA (60 to 200 micrograms/g) than the pars tensa and areas of the medial wall (2 to 7 micrograms/g). In purulent otitis media the pars flaccida contained less HA than in normal ears. The HA concentrations were lower in purulent (3 to 40 micrograms/mL) than in serous (20 to 190 micrograms/mL) effusions. The weight average molecular weight of HA in serous fluid was high (greater than 10(6] and comparable to that in lymph. It is inferred from the study that the subepithelial tissue and its matrix components, eg, HA, must be considered when attempting to understand the function of the middle ear normally and in otitis media.

Hyaluronan content in human inner ear fluids.

Histochemical analyses of the luminal contents of the endolymphatic sac in numerous mammalian species have indicated a presence of acid mucopolysaccharides or proteoglycans. In the present study, one of these substances, hyaluronan (hyaluronic acid; HA), in endolymphatic sac endolymph and vestibular perilymph in humans, was biochemically determined with a highly specific radioassay. A considerable variation in HA concentration was noted between the individual ES endolymph samples, with a mean value of 2.50 micrograms/g (range 0.00-10.86). In perilymph the HA concentrations were less variable and the mean concentration was 0.91 micrograms/g (range 0.56-1.51). The difference between endolymph and perilymph HA concentration was not statistically significant (p greater than 0.05). The ability of HA to interact with proteoglycans, thereby forming giant hydrophilic molecules, could be important for ES function. These molecules may create a swelling pressure which is resistant to compressive forces that under conditions of increased intracranial hydrostatic pressure otherwise could cause ES collapse and impaired function.

Hyaluronan in the rat with special reference to the skin.

The total hyaluronan content has been determined in rats. The animals were frozen and sectioned in a cryostat before digestion with papain and pronase. The hyaluronan content was determined by a specific radioassay and it was found that 250 g rats contained 40-60 mg of the polysaccharide. The recovery from the preparation procedure was close to 100%, as determined from tracer experiments. More than half of the hyaluronan was found in skin, approximately one quarter in the skeleton and supporting structures and less than one tenth in skeletal muscle. Based on calculated lymph flow and lymph concentration of hyaluronan, it seems that a significant fraction of the total hyaluronan in skin (greater than 1%) is removed via the lymphatics in a 24 h period. An attempt was made to isolate undegraded hyaluronan from rat skin by gentle methods giving full recovery in order to estimate the molecular weight of the polysaccharide. Hyaluronan was recovered quantitatively, but as determined from added tracer, it had been degraded. Correction for the estimated degradation gave a molecular weight of several millions for the endogenous hyaluronan.

Hyaluronate in suction blisters from patients with scleroderma and various skin disorders

With a specific radioassay hyaluronic acid (hyaluronate) concentrations have been determined in suction blister fluid from abdominal skin and serum. Healthy subjects, patients with acrosclerosis, CRST, mucinosis and urticaria had 0.8-5.6 micrograms/ml of hyaluronate in their suction blisters, which is about 100 times more than the serum level. Increased concentrations were noted in blister fluid from patients with active lesions of systemic and localized scleroderma as well as lichen sclerosus. Here the increase could be due to an increased production of hyaluronate in the dermis. High levels of hyaluronate were, however, also found in blister fluid from patients with other types of inflamed skin. An increased leakage into the blister of hyaluronate from the dermis, probably through the lymph vessels, therefore seems best to explain the high concentrations of hyaluronate.

Determination of vitamin D in fortified and nonfortified milk powder and infant formula using a specific radioassay after purification by high-performance liquid chromatography

A reliable and sensitive radioassay is described for the determination of vitamin D in milk powder and infant formula. After saponification of the sample interfering compounds like sterols are removed by digitonin precipitation and chromatography on small columns packed with alumina. [3H] Vitamin D is added to the sample as an internal standard, to correct for losses due to previtamin D formation, as well as other procedural losses. Vitamin D is quantitated by a competitive protein binding (CPB) assay after final cleanup of the extract on a straight-phase HPLC column. Diluted sheep serum is used as the source of the binding protein, having equal affinity for both vitamins D2 and D3. With this HPLC-CPB method vitamin D can be determined with high specificity in concentrations as low as 0.1 ng/tube (ca. 0.01 IE/g). Recovery was between 90 and 110%. The coefficients of variation were 2.4 (within run) and 7.5 (between run), respectively.

Concentration and molecular weight of sodium hyaluronate in synovial fluid from patients with rheumatoid arthritis and other arthropathies.

The molecular weight distribution of hyaluronate (HA) in synovial fluid (SF) from 10 patients with rheumatoid arthritis (RA), from six patients with other joint disorders, and from five recently deceased persons without joint affections was investigated by a gel chromatographic procedure. A new and highly specific radioassay was used for determination of the HA concentration in the effluent from the chromatographic column, and this allowed analyses on 0.5 ml or less of untreated synovial fluid. The results confirmed the findings by others that the weight-average molecular weight (Mw) of HA in SF from patients with RA (4.8 X 10(6)) was similar to that in other joint diseases (5.0 X 10(6)) and moderately but significantly (p less than 0.001) lower than that of normal SF (7.0 X 10(6)). Furthermore, the molecular weight distribution of HA in the pathological SF was generally broad and varied considerably between individuals. The HA concentration in the pathological SF varied between 0.17 and 1.32 g/l, which is in accordance with previous reports and considerably lower than that of normal SF. Neither the nature of the arthropathy and the extent of the inflammatory process nor the pharmacological treatment had a tendency to influence the HA concentration in the SF, the mean molecular weight of HA, or its molecular weight distribution. Although the concentration of HA in SF drops in joint disease, the total amount of the polysaccharide is greatly enhanced. Also the amount of high molecular weight polysaccharide (Mw greater than 6 X 10(6)) is in excess in joint disease. The pathological state is therefore characterised not by lack of high molecular weight hyaluronate but by a dilution of it.

All hexokinase isoenzymes coexist in rat hepatocytes.

The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.

Tissue Distribution of Adenosine Diphosphatase Activity in the Rat

The specific activity of adenosine diphosphatase (ADPase) was determined in rat tissue homogenates using a specific radioassay. 14 different tissues were sampled and activity was found in most homogenates examined. The levels ranged from very little activity in the pancreas to high levels in the duodenum and ileum. High levels of ADPase activity found in the gastrointestinal tract could be partially attributed to non-specific alkaline phosphatase. It is concluded that ADPase activity is widely distributed in rat tissues.

The concentration of hyaluronate in amniotic fluid

The concentration of sodium hyaluronate has been determined in amniotic fluid by a specific radioassay. It shows a large individual variation. The mean concentration is approximately 20 μg/ml at Weeks 16–20 of the gestational period. it drops to approximately 1 μg/ml at Week 30 and is then constant until the end of the pregnancy.

Hyaluronate in aqueous humour

The sodium hyaluronate concentration of aqueous humour has been determined in 10–200 μl samples by a specific radioassay. The accuracy of the technique was studied on pooled bovine aqueous humour. Analyses of untreated samples, dialysed samples and samples digested with papain and then dialysed all gave the same results within the error of the method. The mean concentration determined in 22 assays was 4·4±0·2 μg/ml while independent chemical analysis of the cetylpyridinium precipitable material gave a hexuronate content corresponding to 3·9 μg/ml of sodium hyaluronate. Hyaluronidase treatment removed the material reacting in the assay. Addition of exogenous hyaluronate to the aqueous humour could be determined quantitatively. It was concluded that determinations performed on untreated aqueous humour gave valid values of the hyaluronate content in the fluid. The concentration of hyaluronate in bovine aqueous humour increased after death presumably due to a release of hyaluronate from neighbouring tissues. The concentration of hyaluronate in fresh aqueous humour from single eyes (in a few experiments pooled material) was studied in man, cattle, sheep, pig, rabbit, opossum, rat, chicken and mutton bird. Considerable species variation was found (between 0·2 and 4·0 μg/ml). The level in man was found to be 1·1±0·1 μg/ml. Analysis of material from bovine foetuses showed lower levels than in adult animals.

A sensitive, specific radioisotope assay for l-glutamine- d-fructose-6-phosphate aminotransferase

A sensitive and specific radioassay for l-glutamine- d-fructose-6-phosphate aminotransferase (EC 5.3.1.19) activity is presented. Picomoles of product are measurable, and the assay can be applied to systems having limited quantities of available protein, particularly in extracts of either cell or organ cultures. The assay is at least 10,000 times more sensitive under K 1 concentrations of fructose 6-phosphate than the modified Elson-Morgan colorimetric assay and 20 times more sensitive under saturating conditions of fructose 6-phosphate. As little as 0.5 μg of cell-extract protein will yield measurable product. In contrast, 280 μg of crudeextract protein from colon is required with the modified Elson-Morgan colorimetric assay.

Determination of hyaluronate in biological samples by a specific radioassay technique

A radioassay to determine hyaluronate in tissues and body fluids has been developed. 125I-labeled cartilage protein with affinity for hyaluronate was partitioned between hyaluronate in free solution and an agarose gel substituted with hyaluronate. The radioactivity bound to the gel was related to the amount of free hyaluronate in the system. Under the standard conditions selected for the assay, hyaluronate could be determined in the range from 40 to 1000 ng. The standard error in the determination was estimated to be 15–20%. The method has been applied to papain digests of some tissues as well as to some nondigested body fluids. The material reacting in the assay was hyaluronidase sensitive and when exogenous hyaluronate was added to tissue samples the hyaluronate could be recovered in a 80–110% yield, indicating that the assay gives the correct concentration of this polysaccharide in biological material.

Detection and possible functional influence of antibodies directed againt the pancreatic islet cell surface.

Antibodies directed towards determinants on the surface of rat islet cells can be detected qualitatively by the indirect immunofluorescence or quantitatively by a specific radioassay for IgG. Antibodies are found in insulin-dependent diabetics or in rabbits immunized with islet cells. Such antibodies may affect the B-cell function as indicated by the inhibition of incorporation of radioactive leucine into proinsulin/insulin in vitro.