Competitive solid-phase immunoassay of testosterone using time-resolved fluorescence.

A competitive solid-phase immunoassay for the determination of testosterone in serum samples using time-resolved fluorescence is described. The solid phase is a testosterone-3-(O-carboxymethyl)-oxime-ovalbumin conjugate coated to polystyrene microtiter strips. Europium-labelled polyclonal and monoclonal antibodies against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = -0.150 + 0.969x).

Investigation of the fine structure of alpha-dextrins derived from amylopectin and their relation to the structure of waxy-maize starch.

Alpha-dextrins, obtained by fractional precipitation with methanol of the products of the action of Bacillus subtilis alpha-amylase on waxy-maize amylopectin, were debranched with isoamylase and the distributions of the unit chains were analysed by gel-permeation chromatography. The large alpha-dextrins still contained long B-chains after hydrolysis for 60 min, but these were absent from the small dextrins with chain numbers of approximately 11 or less. The small dextrins contained increased amounts of chains with lengths intermediate of those of the long B-chains and the main part of the short chains. After hydrolysis for 210 min, almost all of the long B-chains had disappeared and the chains with intermediate lengths had been shortened further. The distributions of the unit chains of the internal chains, obtained by debranching of the phosphorolysis (phi)-limit dextrins, gave similar results and showed that the ratio of A- to B-chains was unchanged during the alpha-amylolysis. Models for the fine structure of the intermediate alpha-dextrins are proposed.

The substitution pattern in cationised and oxidised potato starch granules.

The distribution pattern of substituents within the granules and the components of two cationised and two oxidised potato starches was studied. The level of crystallinity in wet-cationised (WC) and hypochlorite oxidised (HO) starch granules was similar to that of native starch granules but lower in dry-cationised (DC) and peroxide oxidised (PO) granules. However, the melting temperature of DC granules remained similar to native granules but was decreased in the other samples. With all modified starches, the initial rate of acid hydrolysis (lintnerisation) was increased compared to native granules. The degree of substitution decreased only slightly in WC granules after the lintnerisation, whereas virtually all the substituted glucosyl units in DC starch were hydrolysed already at initial stages. The decrease of substituents in the HO and PO starches was intermediate. The starches were partly resistant to the action of isoamylase and the successive beta-amylolysis, suggesting that substituents were found both close to the branches and near the nonreducing ends in the amylopectin component. It is suggested that the DC starch was preferentially cationised at the surface of the granules, whereas WC and oxidised starches were modified throughout the granules.

Relationship between branching density and crystalline structure of A- and B-type maize mutant starches.

Amylopectin from two double maize mutant starches of A-crystalline (wxdu) and B-crystalline type (aewx) was subjected successively to hydrolysis involving alpha and beta amylases, which isolated clusters and all branching zones of clusters (BZC). Enzymatic analysis together with ionic and size-exclusion chromatography revealed the structural features of the clusters and BZC and their role in starch crystallization. A-type clusters were larger (dp(n) > 80) and contained more (but shorter) chains than B-type clusters. The BZC of A-type starch was also larger, but with a shorter distance between the branching points than in B-type BZC. A-type clusters had a densely packed structure and B-type a poorly branched structure. Models for the structure of A- and B-type clusters are presented, and a hypothesis for the influence of cluster geometry on crystallization is proposed.

Determination of hormones by time-resolved fluoroimmunoassay.

Immunoassays based on europium labels and time-resolved fluorescence as the detection method, have been developed. The specific activity of the label is several orders of magnitude higher than that of radioactive labels. Consequently, the technique provides great potential, especially in the determination of analytes which require high sensitivity. Both competitive and immunometric assays which use labelled antibodies have been worked out. In competitive assays the antigen is immobilized on a solid phase with a protein carrier. The antigen in the standard or sample then competes with the labelled antibody in solution. Separation is done simply by washing the wells in the microtitre strip where the assays are performed. Model systems are described for the measurement of testosterone and cortisol. Immunometric assays of human thyrotropin (hTSH) and luteotropin (LH) were performed with monoclonal antibodies, by either a one-step (hTSH) or two-step (LH) incubation procedure. These assays, which exploit the specific activity of the label, give a very high sensitivity and good reproducibility. The standard curves are linear and the dynamic range is at least 1000-fold. Because of the properties of the europium label and the simple assay design, the immunoassays based on time-resolved fluorescence are expected to gain wide application both in research and in routine determinations.

Enzymic analysis of the structure of oxidized potato starches.

The possibility to use enzymic methods for the analysis of the positions of carboxyl and carbonyl groups in sodium hypochlorite oxidized (HO) and hydrogen peroxide oxidized (PO) potato starches was investigated. The HO-starch, that contained more modified glucosyl residues, possessed a lower beta-amylolysis limit and all of the polymer components were resistant to complete hydrolysis as judged from gel-permeation chromatograms. In contrast, the PO-starch contained 24% of apparently unmodified, linear chains that were hydrolysed by beta-amylase. After debranching, approximately 30% of the chains in the HO-sample and approximately 20% in the PO-sample remained partly resistant to successive beta-amylolysis.

Physiotherapy after fracture of the proximal end of the humerus. Comparison between two methods.

Two methods of physiotherapy after fracture of the proximal end of the humerus were compared. The study was designed as a randomized, controlled and single-blind trial. Twenty patients were assigned to two groups 10-12 days after the injury. One group was treated conventionally, the other received instructions in self-training with follow-up control of results. Objective examination and subjective assessment were made at five intervals, up to one year after the injury. The greatest improvement in function was shown in both groups between 3 and 8 weeks. The patients deemed their daily life functions to be normal 8 weeks after the injury. The objective assessment was normal at later stages. No significant differences were found between the two groups in any of the tests. Instruction in self-training with control of results including objective assessments are therefore an adequate method in the rehabilitation of these patients.

The influence of scapular retraction and protraction on the width of the subacromial space. An MRI study.

The width and configuration of the subacromial space was studied in retraction and protraction of the shoulder girdle in four healthy subjects by magnetic resonance imaging. The anterior opening of the subacromial space narrowed as the shoulder moved from a retracted to a protracted position.

The reliability of three active motor tests used in painful shoulder disorders. Presentation of a method of general applicability for the analysis of reliability in the presence of pain.

This article deals with reliability aspects of standardized, active motor tests ("functional tests") when applied to patients with painful shoulder disorders. Motor performance was rated independently by the same two examiners in a standardized way in three different manoeuvres: the Hand in Neck, Hand in Back, and Pour out of a Pot tests. Pain experienced during these tests was rated by the patients on a verbal scale. A method of general applicability is presented for the analysis of reliability of standardized, active motor tests when applied to painful shoulder joint disorders. The importance of differential motivation is stressed, as is the importance of using reliability measures that are adapted to the specific purpose of a particular clinical investigation.

Pain is a major determinant of impaired performance in standardized active motor tests. A study in patients with fracture of the proximal humerus.

We have followed the recovery of motor performance and the subsidence of pain for one year in 12 patients after fracture of the proximal humerus. Performance scores during standardized active motor tests were recorded 3, 8, 16, 24, and 52 weeks after injury, and the pain ratings during each of three manoeuvres were assessed on a modified Borg verbal scale. The manoeuvres were: Hand in Neck, Hand in Back, and Pour out of a Pot. In a cross-sectional analysis of data obtained 3 weeks after injury, significant correlations were found between movement-induced pain and impairment of performance in all three tests. A multivariate analysis indicated a strong association between decreasing pain and increasing performance and this was significant after elimination of the influence of healing as measured by time. In contrast, the association between time and increasing performance, after eliminating of the influence of decreasing pain, was weak and non-significant. It is concluded that pain is a major determinant of impaired performance after fracture of the proximal humerus, and that performance scores in standardized active motor tests are inversely correlated with the amount of movement-induced pain.

The hand in neck manoeuvre as a tool to analyze pain-generating mechanisms in the subacromial impingement syndrome.

A scoring system for a standardized composite movement of the shoulder--the Hand in Neck (HIN) manoeuvre--is presented. The EMG activity of the supraspinatus muscle was studied in 5 healthy subjects at different performance levels of this test. It is shown that the supraspinatus muscle is about four times more active during normal performance than at the subnormal levels, which among themselves do not differ. EMG activity was also studied during normal performance of another standardized manoeuvre--the Pour out of a Pot (POP) test. Based on a comparison of the EMG data with clinical data from patients with the subacromial impingement syndrome it is suggested that an abnormal HIN test indicates the presence of a traction responsive pain generator in the supraspinatus tendon. In the same patient group, the combination of a normal HIN test and an abnormal POP test indicates pain generated by compression of subacromial structures.

The subacromial impingement syndrome. A study of results of treatment with special emphasis on predictive factors and pain-generating mechanisms.

Forty-two patients with subacromial impingement syndrome entered a randomized prospective study comparing open anterior acromioplasty with a physiotherapy regime. The criterion for a successful outcome of treatment was a reduction of the initial pain score of more than 50% using the visual analogue scale (VAS) technique. The evaluation was performed by an independent observer. At the 6-month follow-up, treatment in the surgical group had been successful in 12/21 (57%) patients versus 6/18 (33%) in the physiotherapy group. A one-year evaluation revealed 16/21 (76%) "successes" in the surgical group. A direct and unbiased comparison with the physiotherapy group was not possible at one year, since 13 patients chose surgery after initial physiotherapy. However, in "an intention to treat" analysis outcome at one year was significantly better in those randomized to surgery. We found two standardized, composite, active movements--the "Pour out of a Pot" manoeuvre requiring the emptying of a pot of water, and the "Hand in Neck" manoeuvre--to be of predictive value for the outcome of surgery. When combining three pain-related variables into a criterion for prediction of success, a sensitivity of 78% (p < 0.02) and a specificity of 90% (p < 0.03) were attained. We advance the hypothesis that pain in the impingement syndrome is mainly elicited by comparison of the subacromial bursa. In some patients a traction-responsive pain generator in the supraspinatus tendon may be present as well.