Serum thyroxine binding capacity-dependent bias in an automated free thyroxine assay.

The magnitude of serum thyroxine (T4) binding capacity (sBC) dependent bias in the AXSYM free thyroxine (FT4) assay was assessed using two recently described tests. One of the tests uses a direct equilibrium dialysis (ED) FT4 assay as the reference method. The results obtained with the AXSYM method were compared with those obtained by the ED FT4 method in patient sera having a wide range of sBC. The other test involves comparison of the FT4 results obtained following dilution of sera by an inert buffer, to theoretically derived FT4 results. As serum dilution causes a predictable decrease in sBC, the demonstration of a negative bias whose magnitude increases in parallel to the dilution, is indicative of an sBC-dependent bias. The AXSYM FT4 assay exhibited a significant sBC-dependent bias. This sBC-dependent bias is likely to have been caused by the presence of significant amounts of T4 binding proteins in the assay reagents.

Assessment of serum thyroxine binding capacity-dependent biases in free thyroxine assays.

BACKGROUND: Free thyroxine (FT4) assays may exhibit biases that are related to serum T4 binding capacity (sBC). We describe two tests that can be used to assess the presence and magnitude of sBC-dependent biases in FT4 assays. METHODS: We used a direct equilibrium dialysis FT4 assay as the reference method and compared the results obtained with those of the FT4 assays under investigation, in patient sera having a wide range of sBC. We then compared the expected and observed FT4 results for sera diluted with an inert buffer. Because serum dilution causes a predictable decrease in sBC, an increasingly negative bias on progressive dilution is indicative of a sBC-dependent bias. RESULTS: The automated FT4 assay investigated (Vitros FT4) showed no demonstrable sBC-dependent bias by either test. CONCLUSION: These two tests can be used to screen for sBC-dependent biases in FT4 assays.

Multicenter evaluation of enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine.

The technical and diagnostic performance of the Amerlite-MAB enhanced luminescence assay for free thyroxine (FT4) was assessed in a multicenter evaluation trial. The euthyroid central 95% reference range for FT4 (1393 subjects) was 11.5-27.7 pmol/L (by cumulative frequency plot with two-tailed 2.5% cutoffs). Results (y) agreed with those of similar radioactive method (Amerlex-MAB FT4) (x): y = 1.06 x + 0.54, Sylx = 335.5, n = 235). Mean within-assay precision (CV) in six centers was 5.7% at 6 pmol/L and 2.6% at 51 pmol/L FT4; between-assay precision (CV) was 7.2% and 3.6%, respectively. Diagnostic performance was assessed in all patient groups usually encountered, including those with nonthyroidal illness and extreme binding-protein anomalies. In elderly euthyroid subjects, the proportion of above-normal FT4 values exceeded that in younger age-groups. These increases often accompanied normal estimates of thyrotropin and, in some cases, might have arisen by interference from medication that is taken more often by the elderly.

Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine: design, development, and technical validation.

We describe the development and validation of a one-step nonradioactive immunoassay for free thyroxine (Amerlite-MAB FT4) in serum or plasma, in the dedicated Amerlite enhanced luminescence assay system. A monoclonal antibody to thyroxine (T4), conjugated with horseradish peroxidase (HRP; EC 1.11.1.7), competes for binding with FT4 or with a conjugate of a protein and L-triiodothyronine immobilized onto the surface of the assay microwells. The signal is generated by antibody-HRP bound to the protein-T3 conjugate, with luminol as substrate. The assay design exploits the high sensitivity of luminescence signal detection, permitting minimal sample dilution and T4 sampling by the antibody. It withstands progressive dilution of serum and is unaffected by T4-binding proteins in serum. The disclosure and validation of this FT4 assay follows guidelines recommended by the American Thyroid Association.

One-step, labeled-antibody assay for measuring free thyroxin. I. Assay development and validation.

We describe a one-step, labeled-antibody radioassay for measuring free thyroxin (FT4) in serum or plasma, based on a novel principle. FT4 in the sample competes with a gross molar excess (over antibody) of a cross-reactant (L-triiodothyronine, T3), chemically coupled to magnetizable polymer particles, for binding to avid 125l-labeled monoclonal anti-thyroxin antibodies. As in conventional immunoassays, 125I counts bound to the solid phase (T3-magnetizable particles) are inversely proportional to sample FT4 concentration. We demonstrate here the development and technical validity of this new method.

One-step, labeled-antibody assay for measuring free thyroxin. II. Performance in a multicenter trial.

The technical and diagnostic performance of a labeled antibody radioassay for free thyroxin (Amerlex-MAB* FT4) was examined in a multicenter trial. The overall mean within-assay precision (CV) in eight centers was 6.2% at 30 pmol/L and 3.6% at 13 pmol/L concentrations of FT4. Between-assay precision was 5.4% at 18 pmol/L and 6.8% at 50 pmol/L. The euthyroid central 95% reference range for FT4 was 11.3 to 24.3 pmol/L. Results of the method correlated well with those of an analog radioimmunoassay: [Amerlex-MAB* FT4] = 1.09 [Amerlex-M FT4] + 1.50 pmol/L (r = 0.96, n = 732). Clinical performance of the assay was better than that of first-generation analog tracer assays for sera from patients with nonthyroidal illness or binding-protein abnormalities.

Differential neuropeptide expression after visceral and somatic nerve injury in the cat and rat.

The expression of neuropeptides galanin, vasoactive intestinal polypeptide (VIP) and substance P was compared after injury to somatic (sciatic, pudendal) and visceral (pelvic) nerves. Studies in normal rats and the mutant rat 'mutilated foot' suggested that galanin increases in sensory but not sympathetic fibres after sciatic nerve injury, while VIP appears to increase in both sensory and sympathetic fibres, and substance P to decrease in sensory fibres. A direct comparison of neuropeptide changes after somatic and visceral nerve injury was made in the cat dorsal sacral spinal cord, where both pudenal (somatic) and pelvic (visceral) afferents terminate. Four weeks after pudendal nerve transection in the cat there was an increase of VIP and galanin but decrease of substance P in the dorsal sacral cord, similar to the changes in lumbar dorsal cord after sciatic nerve section in the rat. In contrast, 4 weeks after pelvic nerve transection in the cat, galanin was unchanged in the ipsilateral dorsal sacral spinal cord, whereas VIP is known to decrease markedly and substance P to remain unchanged. There is thus differential peptide expression before and after injury in somatic and visceral systems, which may be regulated in part by the target organ. We have proposed that the neuropeptide changes occur in neurons that regulate development, maintenance and repair after injury, processes that may differ in somatic and visceral systems.

Concentrations of free thyroxin and albumin in serum in severe nonthyroidal illness: assay artefacts and physiological influences.

Free thyroxin (FT4) estimates by two immunoassays were compared with the concentrations of albumin in serum of apparently euthyroid subjects who either were (n = 99) or were not (n = 327) suffering from severe nonthyroidal illness (sNTI). In neither group was FT4 significantly correlated with albumin (P greater than 0.05), according to a "labeled antibody" radioassay (Amerlex-MAB). On amalgamating both groups, correlation with albumin was positive and significant (P less than 0.001). In the group with sNTI, both FT4 and albumin concentrations were decreased (mean FT4 to 77% and mean albumin to 61% of the respective reference means). For an analog radioimmunoassay (Amerlex-M), FT4 in all groups was significantly (P less than 0.001) correlated with albumin. Correlation coefficients were greater than with Amerlex-MAB for both sNTI and euthyroid groups, as well as for the joint panel. Mean FT4 in sNTI was only 44% of the reference mean. Lower radio-tracer "analog" values in sNTI are exaggerated by additional technical artefacts resulting from tracer binding to albumin.

Distribution of a novel pituitary protein (7B2) in mammalian gastrointestinal tract and pancreas.

The distribution of a novel pituitary protein (7B2) was determined in the gastrointestinal tract and pancreas of four mammalian species (man, pig, guinea pig, and rat) by a specific radioimmunoassay. The highest concentrations of cross-reacting immunoreactive 7B2 (IR-7B2) were observed in the pancreas and the proximal gut (antrum or duodenum). While the intestinal concentrations varied widely among species, pancreatic IR-7B2 concentrations appeared to be similar in all four species. In the rat, pancreatic islets were found to contain high concentrations of IR-7B2 (5.73 +/- 0.14 fmol/islet, mean +/- SEM). Neonatal capsaicin treatment and enteric nerve section did not affect the concentrations of IR-7B2 in the rat intestine. Layer separation of human gut showed that IR-7B2 is mainly (71 +/- 8%) present in the epithelial fraction. Chromatographic analysis of intestinal and pancreatic extracts from the four species on Sephadex G-100 showed the presence of two immunoreactive peaks at Kav 0.3 and 0.6, but there were both inter- and intraspecies variations in the proportions of the larger and smaller molecular forms.

Developmental changes in immunoreactive content of novel pituitary protein 7B2 in human pancreas and its identification in pancreatic tumors.

Developmental changes in concentration of a novel pituitary protein (7B2) were studied immunochemically in the human pancreas from 20 wk of gestation to 4 mo after birth. The concentrations of 7B2 in pancreatic islet tumors and in nesidioblastosis were investigated also. Significant quantities of 7B2-like immunoreactivity (IR-7B2) were found in all developmental stages studied. The highest concentrations of IR-7B2 were found at term [215.4 +/- 40.0 vs. 28.3 +/- 4.4 pmol/g (adult levels), P less than .001]. A high incidence of elevated IR-7B2 concentration in pancreatic islet tumors and nesidioblastosis was found (10 of 12 insulinomas, 5 of 8 glucagonomas, and in all 3 pancreases with nesidioblastosis). Gel-permeation chromatography on Sephadex G-100 showed two immunoreactive peaks in all extracts studied. The main peak (Kav 0.30) of IR-7B2 corresponded to that found in the porcine pituitary gland. The high incidence of elevated IR-7B2 concentrations in pancreatic islet tumors and the increase in IR-7B2 concentrations in the term pancreas and particularly in nesidioblastosis suggest that the novel protein 7B2 may serve as an islet marker.

Effect of naloxone on the antral motor response to solid food in man.

Antroduodenal motor activity was recorded in eight healthy subjects using perfused tubes connected to external strain gauge transducers. Each subject was studied over a 2.5-h period following ingestion of a solid meal, on 2 separate days. Intravenous saline was administered on one day and saline plus naloxone (40 micrograms kg-1 h-1) on the other, in randomized order. Naloxone markedly inhibited the antral motor response to food and this effect was due to decreased amplitude and contractile frequency. The duodenal motor response to solid food and the postprandial rise in serum gastrin and plasma pancreatic polypeptide were not altered by naloxone. These observations suggest that peripheral or central opiate receptors play a role in regulating the antral motor response to food.

Resolution of acromegaly after removal of a bronchial carcinoid shown to secrete growth hormone releasing factor.

A 29 year old woman with an enlarged pituitary fossa and classical acromegaly, possibly present for ten years, had biochemical and partial somatic resolution of the disorder after removal of a bronchial carcinoid tumour. In addition, galactorrhea stopped, menstruation returned after two years, and amenorrhea and elevated prolactin levels fell towards normal. Immunocytochemistry showed numerous growth hormone releasing factor (GRF) staining cells in the tumour. The tumour cells, when cultured, produced a supernatant selectivity stimulating human pituitary somatotrophic cell cultures to produce growth hormone (GH). The bronchial carcinoid did not secrete detectable GH, but extracts of it, and preoperative serum contained GRF immunoreactivity which coeluted with synthetic human pancreatic GRF.

Motilin secretion and the migrating myoelectric complex in the pig.

The concentration of motilin in plasma from the abdominal aorta and the hepatic portal vein and the net portal motilin output varied with the phase of the migrating myoelectric complex (m.m.c.) in five of six pigs fasted for 17 h. Maximum concentrations and output occurred 9-12 min before phase III in the duodenum or upper jejunum. In fed pigs m.m.c.s occurred and the first phase III in the duodenum occurred within 90 min of feeding. Both portal and arterial motilin concentrations were reduced after feeding and no longer varied with the phase of the m.m.c. Altered secretion of motilin after feeding did not appear to be associated with absorption of glucose as infusion of glucose (50 g/l, 10 ml/min) into the duodenum raised arterial and portal plasma glucose concentrations to post-prandial levels yet motilin concentrations and output rates still varied with the phase of the m.m.c. Infusions of motilin (1 or 10 ng/kg X min) into the portal vein of 17 h fasted pigs did not induce an extra phase III or alter the duration of the m.m.c. Hydrochloric acid (100 mmol/l) infused into the duodenum of fasted pigs at 10-21 ml/min increased the concentration of motilin in the portal blood but was without effect at 5 ml/min. Rapid injections of 50 ml hydrochloric acid into the duodenum also increased the portal motilin concentration. Hydrochloric acid infusion or injection did not alter the interval between phase IIIs. It is concluded that motilin secretion is a consequence of the m.m.c. or shows the same periodicity as the m.m.c. but that motilin is not an important factor in the initiation and control of the m.m.c. in the pig.

Distribution and molecular heterogeneity of galanin in human, pig, guinea pig, and rat gastrointestinal tracts.

Galanin was measured by radioimmunoassay in whole thickness extracts of the gastrointestinal wall from four species and in extracts from separate layers of human small intestine. The immunoreactivity was characterized using gel chromatography and high-pressure liquid chromatography. Two antibodies were employed, which were characterized as non-C-terminal (Gal 8) and C-terminal (Gal 9) using a C-terminal galanin 10-29 fragment. Substantial quantities of galanin immunoreactivity were found, mainly localized at the muscle layer. Both intramolecular and intermolecular heterogeneity was apparent. Two molecular forms exist in humans (Kav 0.58, 0.69). The molecular heterogeneity in humans, rats, and guinea pigs may be localized near the C-terminus of the galanin molecule. A C-terminal extension of one human galanin form is likely (Kav 0.58). These findings give radioimmunologic evidence for a neurocrine origin of galanin. The chromatographic variations suggest that extrapolation of experimental results between species should be treated with caution.

Occurrence and distribution of a newly discovered peptide, galanin, in the mammalian enteric nervous system.

Galanin, a newly discovered peptide, was found throughout the gastrointestinal tract of man, pig, and rat, exclusively in nerves. The concentrations of immunoreactive galanin ranged from 3.7 +/- 0.7 (mean +/- SEM) pmol/g in rat antrum to 76.5 +/- 14.3 pmol/g in pig colon. The predominantly intrinsic origin of the galanin nerves was shown by the finding of the peptide in submucosal ganglion cells, the majority of which also contained VIP. Furthermore, neither extrinsic denervation of the gut nor administration of capsaicin, which selectively destroys extrinsic afferent fibres, had any significant effect on the galanin innervation. The caudal projection of galanin-immunoreactive fibres was demonstrated by complete transection of the gut, which led to their reduction in the 1 to 2 cm distal to the cut. The abundance of galanin in the innervation of the mammalian gut and its reported action on smooth muscle contractility suggest this peptide to be a novel regulatory factor in the control of bowel function.

Plasma release of atrial natriuretic peptide in response to blood volume expansion.

The response of plasma atrial natriuretic peptide (ANP) concentration to acute intravascular volume expansion was measured in ten male Wistar rats. An infusion of 3 ml polygelene colloidal solution at 37 degrees C over 45 s produced peak venous pressure rises of 1.5 cm water. A highly significant (P less than 0.001) rise of immunoreactive plasma ANP from 24.4 +/- 2.2 (mean +/- S.E.M.) pmol/l to a peak of 70.0 +/- 10.5 pmol/l occurred within 2.5 min. Plasma ANP concentrations had virtually returned to basal levels (32.7 +/- 2.7 pmol/l) 30 min after this acute volume load. A further infusion of 10 ml polygelene colloidal solution in 2 min produced peak venous pressure rises of 10 cm water and caused a dramatic and significant (P less than 0.001) increase of plasma ANP concentration to a peak of 534.8 +/- 38.5 pmol/l, occurring 7.5 min after infusion. The plasma ANP concentration had fallen but remained above basal levels 30 min later (137.2 +/- 26.4 pmol/l). Similar results were obtained using an identical protocol but with whole rat blood instead of polygelene solution as the volume-expanding agent. Gel column chromatography suggested that the majority of the immunoreactive ANP in rat plasma was of similar molecular size to rat alpha-ANP (1-28). These results support the hypothesis that blood volume expansion is a potent stimulus for the release of ANP into plasma.

Radioimmunoassay of alpha rat atrial natriuretic peptide.

Specific antibodies to alpha 1-28 atrial natriuretic peptide have been raised and used for radioimmunoassay of tissue extracts and for light and electron microscopic immunocytochemistry. The radioimmunoassay has been used to quantitate ANP-immunoreactivity in normal rat heart and hypothalamus and immunocytochemistry to demonstrate its localisation in specific tissue structures. Gel chromatography and high pressure liquid chromatography confirm that the majority of ANP-like immunoreactivity in the atria exists as high molecular weight forms. Rat hypothalamus contains immunoreactive ANP; the concentration per gram of tissue being 2-4 thousand fold less than that of the cardiac atria. The supraoptic region of the hypothalamus did not have a significantly different concentration of ANP-like immunoreactivity from the hypothalamic region as a whole. Immunocytochemical staining with ANP antiserum revealed the cardiac ANP-immunoreactivity to be concentrated around the nuclear poles within the cytoplasm of atrial muscle cells. Electron microscopic study of atrial cells stained with the immunogold technique confirmed the localisation of the ANP immunoreactivity to electron-dense secretory granules. The highest density of regional immunoreactive staining was in the subepicardial area, the lowest in the interatrial septum. The finding that the highest quantities of ANP immunoreactivity occur in areas subjected to the greatest distensional forces supports the hypothesis that atrial stretch is a stimulus to the release of this peptide from cells.

A novel pituitary protein (7B2)-like immunoreactivity is secreted by a rat phaeochromocytoma cell line (PC12).

High concentrations of a novel pituitary protein (7B2) have been shown to be present in the PC12 rat phaeochromocytoma cell line by radioimmunoassay. 7B2-like immunoreactivity (IR-7B2) was released from PC12 cells into the incubation medium in response to stimulation by a depolarizing concentration of K+, and this K+-evoked release was inhibited by Co2+. The major IR-7B2 in PC12 cell and medium appeared to be identical to that in porcine pituitary gland as judged by both gel permeation chromatography and by reverse-phase high performance liquid chromatography (HPLC). Gel permeation chromatography of extracts of cell and medium revealed two IR-7B2 peaks, the earlier eluting at a elution coefficient (Kav) of 0.30 and the later at a Kav of 0.54. In medium, over 90% of the IR-7B2 eluted as the earlier peak. Fractionation of extracts of cell and medium on reverse-phase HPLC showed three main IR-7B2 peaks eluting at 43, 44.5 and 46% acetonitrile/water with 0.1% trifluoroacetic acid. The findings suggest that IR-7B2 might be released by calcium-mediated exocytosis.

Distribution of galanin immunoreactivity in the genitourinary tract of man and rat.

Galanin has been shown to be present in substantial quantities in the human and rat genitourinary tract by radioimmunoassay and immunocytochemistry. The highest concentrations measured by radioimmunoassay were found in the human vas deferens, corpus cavernosum and spongiosum and in the vagina and cervix. In man gel chromatographic analysis showed two molecular forms. The earlier eluting peak was different from porcine galanin standard. There was only one molecular form in the rat which emerged in an earlier position than the porcine standard. Galanin immunoreactive nerve fibres demonstrated in the genitourinary tract were found both in man and rat. They were found within smooth muscle and in close relationship to blood vessels. The presence and distribution of galanin in the genitourinary system suggest the possibility that this neuropeptide could play a role in the regulation of smooth muscle tone, blood flow and motility.