Detection of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in beer and wine

A carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was measured in beer and wine by HPLC. PhIP was found to be present in all brands of beer and wine analyzed. The concentrations of PhIP in beer and wine were 14.1 +/- 6.18 ng/l (mean +/- SD, n = 11) and 30.4 +/- 16.4 ng/l (n = 10) respectively.     

Modified radioiodination and quality control methods for [125I]sodium iothalamate

[125I]Sodium iothalamate can be prepared by the isotope-exchange method with the use of a contrast medium preparation (i.e. iothalamate sodium injection, USP, 80%). The initial isolation and purification of iothalamate from the contrast medium solution for radioiodination is tedious and time-consuming (i.e. 1 1/2 h for purification and overnight for drying). The new method uses iothalamic acid to replace iothalamate sodium injection as a starting material and reduces the heating time and multiple acid-washing steps during radioiodination to expedite the radiolabelling process. The radiochemical purity (RCP) of [125I]sodium iothalamate obtained from the new method was 98.9 +/- 1.3% (n = 30) versus RCP value of 99.2 +/- 1.0% (n = 25) from the old method with no significant differences between the two groups of RCP values. An RCP chromatographical system to separate and migrate the radiochemical species of [125I]sodium iothalamate from the origin is described in this paper.     

A simple method for measurement of angiotensin II in plasma

A simple radioimmunological (RIA) method for the determination of angiotensin II in 0.5-1.0 ml samples of plasma is described and carefully evaluated. Before RIA was performed, the interfering plasma proteins were eliminated by ion exchange chromatography, and recovery from every column was checked with a small amount of [125I]angiotensin II. The sensitivity of the method was 4.0 ng/l; the coefficient of intra-assay variation was 10.0% and that of inter-assay variation 12.1%. Accuracy was studied both by adding various amounts of angiotensin II to plasma samples and by diluting plasma containing angiotensin II with the RIA buffer. Both studies gave very good correlations between found and expected values (r=0.998 and r=0.987). In a normal material (n=36), the mean angiotensin II concentration at 8 a.m. after 2 h ambulation 42.4 +/- 12.8 (S.D.) ng/l. Because the present method is accurate, precise, and practical, and allows measurement of angiotensin II in small samples, it seems useful for routine as well as for research work.     

A simple and rapid thyroxine radioimmunoassy (T4-RIA) in unextracted human serum; a comparison of T4-RIA and T4 displacement assay, T4(D), in normal and pathologic sera

A simple, rapid and accurate thyroxine radioimmunoassay (T4-RIA) in unextracted serum or plasma has been described, and for comparison T4 determinations have also been made by a T4(D) procedure using Abbott Tetrasorb kits. T4-RIA procedure basically involved denaturation of serum to dissociate T4-protein bond, and T4 released was allowed to react with [125I]T4-labeled T4 antiserum elicited by immunizing rabbits against bovine thyroglobulin. The displaced unbound [125I]T4 was rapidly taken up by an anionic resin sponge within 15 min and this sponge [125I]T4 uptake was linearly related to T4 present in standards or serum. The denaturation of serum effected by trichloroacetic acid-sodium hydroxide permitted virtually 100% T4 extraction recovery in normal, pregnancy, hypo- and hyperthyroid sera whereas 72.9-87.6% T4 recovery from normal serum (and with large individual differences) was noted with lower alcohols in T4(D) procedure. Cumbersome and/or tedious steps such as pre-extraction, centrifugation, time consuming bound and unbound hormone separation procedures, etc. are obviated in T4-RIA and the entire assay can be completed in the same tube in approximately an hour. These attributes along with increased sensitivity and specificity and the need for only microamounts of test sera (25-50 mul) in T4-RIA offer distinct advantages over T4(D) procedures, and in simplicity excel even other T4-RIAs. T4-RIA values in physiological and pathological states were highly correlated (r = 0.97) with T4(D) measurements and no differences between these two techniques were found. The reported discrepancies between T4-RIA and T4(D) measurements in human sera and some of the reasons for attributing these inconsistencies to probable methodological errors and variations are discussed.     

Radioimmunossay of secretin in human plasma

A radioimmunoassay is presented which employs 125I-labelled synthetic secretin, antibody against synthetic secretin, and standards prepared from pure natural porcine secretin. Secretin to be measured was extracted into methanol from heparinized plasma containing aprotinin, which together with cysteine hydrochloride was used as stabilizer throughout the assay. With polyethylene glycol separation, a within assay precision of 10% at 17 pmol/1 was found. The between assay precision was 15% at 17 pmol/1 and thelimit of detection 2.5 pmol/1 plasma. Accuracy was 70-85%. The immunoreactive secretin levels in human plasma increased from 4.5+/-0.5 pmol/1 (mean+/-S.E.M.) to 19.5+/-7.5 pmol/1 (mean+/-S.E.M.) after duodenal acidification (n=5). Pancreatic flow rate increased from 0.5+/-0.1 ml/min (mean+/-S.E.M.) to 4.8+/-0.5 ml/min (mean+/-S.E.M.), and bicarbonate output from 9.6+/-1.8 mumol/min (mean+/-S.E.M.) to 268+/-51 mumol/min (mean+/-S.E.M.) after duodenal acidification.     

Determination of free follistatin levels in sera of normal subjects and patients with various diseases

We developed an assay system for measuring free follistatin by using an anti-follistatin mouse monoclonal antibody and [125I]activin A. The sensitivity of this assay was 0.5 microgram/l and cross-reactivities with inhibin, luteinizing hormone, follicle-stimulating hormone and growth hormone were all less than 0.5%. The dose-response curves of human sera and follicular fluid were parallel to the standard curve, and the follicular fluid contained a large amount of follistatin (6.4 +/- 0.5 mg/l, mean +/- SEM; N = 13). The within- and between-assay coefficients of variation calculated from the analysis of serum samples of four different concentrations were 3.3-7.8% and 3.9-11.0%, respectively. The recovery rates of free follistatin at five different doses were 86.4 - 102.4%. When activin A was added to the same sample, free follistatin recovery rate declined dose-dependently. Gel filtration analyses of human serum and follicular fluid resulted in a single peak corresponding to authentic follistatin. Using this assay, free follistatin concentrations in sera were measured in normal, pregnant and diseased subjects. The free follistatin level in serum of normal adults was 3.5 +/- 0.2 micrograms/l (N = 60), which was significantly elevated in pregnant women (16.7 +/- 1.3 micrograms/l, N = 56), and in patients with chronic liver disease (8.1 +/- 1.1 micrograms/l, N = 20), chronic renal failure (6.7 +/- 0.9 micrograms/l, N = 42), advanced solid cancer (8.5 +/- 1.0 micrograms/l, N = 39) and hematological malignancies (6.8 +/- 1.0 micrograms/l, N = 18). These data indicated that the free follistatin concentration in serum is detectable and varies during pregnancy and in various diseased states.     

Reduced variation of tracer binding in digoxin radioimmunoassay by use of (125I)-labeled tyrosine-methyl-ester derivative: relation of thyroxine concentration to binding

Between-sample variation in tracer binding in the 125I-labeled digoxin radioimmunoassay was investigated with two tracers, 3-O-succinyl-digoxigenin-[125I]-labeled tyrosine and [125I]-labeled tyrosine-methyl-ester-digoxin. Digoxin-free serum samples having various concentrations of thyroxine were assayed with both tracers. The percentage of tracer bound when the samples were assayed with the first-mentioned tracer was increased significantly for the low thyroxine groups when compared to the normal (P less than 0.001) or the high thyroxine groups (P less than 0.05). Little difference existed when the latter tracer was used. There was variation in tracer binding when serum from dogs dosed with thyrotropin was assayed with the first tracer, but there was little variation with the second. Tracer binding may be influenced by thyroxine-binding proteins. Variation in tracer binding appears to be reduced when [125I]-labeled tyrosine-methyl-ester-digoxin is used.     

Nitric oxide: description of a pipeline delivery system

The effect of azelastine hydrochloride (azelastine) on synthesis and release of platelet activating factor (PAF) in alveolar macrophages obtained from asthmatic and non-asthmatic subjects was examined. Alveolar macrophages (AMs) were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 microM) for 15 min. PAF activity was detected by aggregation of washed guinea pig platelets. PAF activity released from alveolar macrophages (AMs) from asthmatics without preincubation of azelastine was 15.97 [2.17] (mean [SD], ng/10(7) cells) in supernatants and 42.52 [10.16] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF activity reduced to 10.71 [2.73] (mean [SD], ng/10(7) cells), 7.86 [0.94], and 3.52 [0.31] in the supernatants, and 35.58 [7.37], 21.57 [4.36], and 14.77 [0.99] (n = 15) in the cell pellets, respectively. PAF activity in non-asthmatic subjects without preincubation of azelastine was 8.55 [1.16] (mean [SD], ng/10(7) cells) in supernatants and 32.64 [3.37] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF activity reduced to 6.68 [0.78] (mean [SD], ng/10(7) cells), 4.47 [0.51], and 2.97 [0.36] in the supernatants, and 29.53 [3.75], 14.78 [1.95], and 6.16 [0.55] (n = 20) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra- and extracellular PAF activity from asthmatic and non-asthmatic macrophages in the same manner.     

Radioimmunoassay of glutathione peroxidase in human serum

Glutathione peroxidase (GSHPx) was purified from human serum and used for immunization of rabbits. Antiserum bound up to 75% of added 125I-GSHPx after precipitation with a second antibody. Human serum, but not sera from eight animal species, inhibited the binding of labelled GSHPx, indicating that the antiserum did not react with GSHPx from these species. GSHPx could be measured in less than 10 microliters of human serum by radioimmunoassay. In sera with widely varying selenium concentrations (0.1-2.9 mumol/l) the amount of GSHPx protein (0.3-6.3 mg/l) was strongly correlated with GSHPx activity (r = 0.94) and it was also correlated with serum selenium concentrations (r = 0.64). This indicates that GSHPx protein may be a valuable biological marker of selenium status. In samples with serum selenium concentrations of 0.8-1.2 mumol/l, the concentration of GSHPx was 3.3 (0.4) mg/l (mean (S.D.)), or 0.04 (0.005) mumol/l. This corresponded to 0.16 (0.02) mumol/l of GSHPx selenium and 16% (2.8)% of total serum selenium. The data suggest that the method can be used to measure the proportion of serum selenium that is located in GSHPx. Following storage of serum at room temperature, both serum GSHPx protein and activity declined, but addition of glutathione protected both GSHPx protein and activity.     

Accelerated evolution of cytochrome b in simian primates: adaptive evolution in concert with other mitochondrial proteins?

We have devised a practical, sensitive and specific method for simultaneous measurement of free thyroxine (FT4) and free triiodothyronine (FT3) in undiluted serum by direct equilibrium dialysis radioimmunoassay (RIA). Two hundred microliters serum sample was dialyzed against buffer (pH 7.4) for 20 hours at 37 degrees C and approximately 800 microL of the dialysate was used for measuring FT4 and FT3 simultaneously. The assay was set up in polystyrene tubes coated with anti-T4 antibody and available commercially for FT4 measurement (Quest-Nichols Institute, San Juan Capistrano, CA). The mean +/- SE (range) FT4 concentration (ng/dL) was 1.2 +/- 0.04 (0.7.0 to 2.30) in 54 normal subjects. It was significantly increased (3.6 +/- 0.4 [1.8 to 9.6], n = 20) in hyperthyroidism and clearly decreased (0.40 +/- 0.04 [1.10 to 0.70], n = 26] in hypothyroidism. All nonthyroid illness (NTI) patients had normal FT4 except 3, 2 of whom were on amiodarone and 1 had received heparin. Serum FT4 concentration was minimally elevated in 18 newborn cord blood serum (1.40 +/- 0.08 [0.90 to 2.2], cf. normal p < .05). The mean serum FT3 concentration (pg/dL) was 285 +/- 10 (134 to 454) in 54 normal sera. It was clearly increased in hyperthyroidism (1033 +/- 98 [593 to 2134], n = 20, p < .001). However, serum FT3 varied widely in hypothyroidism (27 to 597, mean 235 +/- 24, NS) as did serum total T3 (19 to 175). Interestingly, however, the mean serum FT3 concentration was normal (273 +/- 28 [62 to 575, NS]) in 25 NTI patients. All of these patients had low serum total T3 (46 +/- 5.0 [10 to 84], ng/dL; normal 84 to 160, p < 0.001), while FT3 was clearly normal in 21 of 25 patients and low in the remaining 4 patients. Similarly, among 18 newborn cord blood sera serum FT3 concentration was normal in 15 and subnormal only in the remaining 3 while all had clearly subnormal total T3 (28 to 74 ng/dL). CONCLUSIONS: (1) A practical, sensitive, and specific assay for simultaneous measurement of FT4 and FT3 is described; (2) FT3 is consistently elevated in hyperthyroidism while FT4 is elevated in most (approximately 85%) cases; (3) FT4 is consistently decreased in hypothyroidism but FT3 varies widely; (4). Serum FT3 concentration is normal in approximately 83% of patients with the low T3 syndrome in NTI and newborn cord blood serum. These data suggest that normal FT3 may explain clinical euthyroidism in many patients with the low T3 syndrome.     

Nitric oxide: a biological effector. Detection using electron paramagnetic resonance

The synthesis and preliminary biological characterization of two isomeric technetium labeled complexes (2,5,5,9-tetramethyl-4,7-diaza-7-(3' (R)-quinuclidinylcarboxymethyl)-2,9-decanedithiolato oxo 99/99mtechnetium(V), [99/99mTc]-1 and [99/99mTc]-2) designed to exhibit affinity to muscarinic cholinergic receptors are described. In vitro binding assays were conducted in mouse brain homogenates (whole brain-cerebellum) at 37 degrees C by the centrifugation method, where non-specific binding was defined by atropine (1 microM). The measured affinity (KD) of [99Tc]-1 for mAChR was 1.9 +/- 0.5 microM (mean +/- SEM; n = 3) and [99Tc]-2 was 4.5 +/- 0.5 microM (mean +/- SEM; n = 3). Scatchard analysis indicated that Bmax values were 10.6 +/- 0.5 and 16.9 +/- 0.5 pmol/mg tissue, respectively. In competition assays, [99Tc]-1 exhibited an apparent affinity (KI) of 16.5 microM (n = 2) against [125I] iododexetimide, whereas [99Tc]-2 exhibited an affinity (KI) of 105 microM. In vivo, 0.3% of the injected dose of [99mTc]-1 and [99mTc]-2 accumulated in the brain at 5 min after injection. These values indicate technetium analogues of neuroreceptor binding ligands can be synthesized and retain some affinity for the receptor.     

Calcitriol modulates in vivo and in vitro cytokine production: a role for intracellular calcium

Calcitriol modulates in vivoand in vitro cytokine production: A role for intracellular calcium. Background. Several immunomodulatory properties of calcitriol are currently known, however, only little information is available regarding the in vivo and in vitro effects of calcitriol on cytokine production in chronic renal failure. Methods. To study the in vitro effect of calcitriol on lipopolysaccharide (LPS)-induced cytokine production, peripheral blood mononuclear cells (PBMC, 2.5 ml/ml) from 12 chronic dialytic (HD), 15 undialyzed chronic renal failure (CRF) patients and 10 normal subjects (N) were incubated at 37 degrees for 12 hours with 100 ng of LPS (E. coli and P. maltofilia). Increasing doses of calcitriol from 10-10 to 10-9 M were added and cell associated TNF-alpha and IL-1beta were determined by immunoreactive tests after three freeze-thaw cycles. The intradialytic TNF-alpha and IL-1beta production were evaluated in vivo in 12 HD patients before and after three months of intravenous calcitriol treatment (6 microgram/week). Intracellular calcium [Ca++]i was determined on PBMC with a cytofluorimetric assay using FLUO-3 AM as the indicator. Results. In vitro, TNF-alpha increased from 3.6 +/- 1.9 pg/cell to 1797 +/- 337 in N, from 4.5 +/- 1.7 to 1724 +/- 232 in CRF and from 3.4 +/- 2.3 to 1244 +/- 553 in HD after the LPS stimulus. The production of TNF-alpha was inhibited by calcitriol in a dose-dependent manner [LPS + Vit.D3 100 ng, 2.9 +/- 2.1 in N, 3.7 +/- 1.9 in CRF and 3.4 +/- 1.7 in HD; LPS + Vit.D3 50 ng, 263 +/- 296 (N), 6.73 +/- 11 (CRF), 38 +/- 28 (HD); LPS + Vit.D3 25 ng = 873 +/- 583 (N), 325 +/- 483 (CRF), 588 +/- 507 (HD); LPS + Vit.D3 12.5 ng, 954 +/- 483 (N), 912 +/- 510 (CRF), 875 +/- 527 (HD)]. Comparable data were observed on IL-1beta production. In vivo, the intradialytic TNF-alpha increase (from 8.5 +/- 2.3 to 19 +/- 5.6 pg/2.5 x 106 cell) during hemodialysis was markedly reduced after calcitriol therapy (from 6.6 +/- 3.1 to 11 +/- 4.7). [Ca++]i decreased from 105 +/- 25 to 72 +/- 18 nM (P < 0.05) and a positive correlation between cytokine levels and [Ca++]i was found (r = 0.79; P < 0.001). Conclusions. The in vitro increase of cell-associated cytokine after LPS challenge was inhibited by calcitriol in a dose-dependent manner. These data suggest a possible in vivo modulatory effect of calcitriol therapy on cytokine production in hemodialysis.     

Radioimmunoassay of 8-arginine-vasopressin (antidiuretic hormone) in human plasma

A radioimmunoassay for 8-arginine-vasopressin (AVP) measurement in human plasma has been developed and evaluated, using a commercial preparation of an antibody of AVP. Detection limit of the assay was 0.4 pg. A simple acetone extraction procedure gave a recovery of 65% of added [125I]AVP. The overall sensitivity in the assay was 1.0 pg/ml when 2 ml plasma samples were extracted. The antigenic sites of the employed antibody seemed to be a combination of amino acid residues in the tripeptide tail and the pentapeptide ring. This can explain that the antibody was almost completely insensitive to chemically or enzymatically degraded AVP. The inter-assay coefficient of variation for the control plasma pools averaged 17%. A good correlation to plasma osmolalities above 290 has been found. AVP level in recumbent subjects (n = 8) with plasma osmolalities in the normal range was 2.8 +/- 1.0 pg/ml (mean +/- SD) and in ambulatory subjects (n = 10) on ad lib. water intake 4.5 +/- 1.9 pg/ml (mean /+- SD).     

Redioimmunoassay for secretin (author's transl)

The synthesis of a secretin with an elongated N-terminus, namely Nalpha-(deaminotyrosyl-beta-alanyl)- secretin (DATA-secretin), using a conventional strategy, is described. After purification of the product by means of ion exchange chromatography on SP-Sephadex C-25 and by continuous carrier-free electrophoresis, immunological studies on the synthetic DATA-secretin were carried out using secretin antibodies. In comparison to 125iodine-labelled secretin and [Tyr6]secretin, 125iodine-DATA-secretin proved to be by far the best "tracer". Elaboration of a radioimmunoassay for secretin is therefore possible.     

Charcot spinal arthropathy in congenital insensitivity to pain

Regioselective radioiodination of N-trifluoroacetyl 3,4-dimethoxy-6-trifluoroacetoxymercurio-L-phenylalanine ethyl ester 1 under no-carrier-added condition gave 6-[125I]iodo protected L-DOPA with a labeling efficiency of more than 85%, and no-carrier-added 6-[125I]I-L-DOPA was obtained with a radio-chemical purity of over 95% after hydrolysis and chromatography. A nonradioactive standard of 6-iodo protected L-DOPA was synthesized by the iododemercuration of 6-mercuric trifluoroacetate protected L-DOPA 1 using I2 in chloroform. 6-[125I]I-L-DOPA showed high brain accumulation and rapid blood clearance in mice. The rat brain slice studies indicated high affinity of 6-[125I]I-L-DOPA for carrier-mediated and stereoselective active transport systems. The tissue homogenate analysis revealed that most of the accumulated radioactivity was intact 6-[125I]I-L-DOPA. Thus, 6-[123I]I-L-DOPA appears to be a suitable single photon emission computed tomography (SPECT) tracer for the selective measurement of cerebral L-amino acid transport, having no affinity for dopamine metabolism.     

Angiography in coxarthrosis (author''s transl)

The preparation of 125I-labelled tracer for digoxin radioimmunoassay (125I monoiodinated 3-succinyl digoxigenin-1-tyrosine) is described, and its performance in radioimmunoassay of plasma samples is compared with that obtained with tritiated digoxin. The accuracy levels were assessed through the evaluation of different potential sources of systematic errors, such as interference from digoxin-related molecules and plasma proteins and methodological artefacts possibly associated with the immunocomplex instability, and through a series of checks including the recovery and parallelism tests and the correspondence of results obtained with the two tracers. The slope and the repeatability with time of the calibration curves and the spread of replicate estimates were taken into consideration to assess the assay precision. An essential equivalence in terms of reliability of measurement was proved for the two methodological variants, so that practical aspects and economic factors remain the main criteria to evaluate the relative merits: from this point of view, the advantages of using 125I-labelled tracer, as an alternative to tritiated digoxin, are discussed.     

Reciprocal effects of thyroxine and triiodothyronine on their deiodination by rat tissues in vitro

The reciprocal effects of loading doses of thyroxine (T4) or triiodothyronine (T3) on the deiodination of their 125iodine-labelled isotopes by rat muscle and liver homogenates were studied. In 21 experiments muscle homogenates deiodinated a mean 45.0% of a tracer dose of [125I]T4 and 18.0% of [125I]T3. On addition of graded amounts of nonradioactive T4 or T3 the percentual deiodination of both labelled hormones progressively declined. This effect was significantly greater in homogenates incubated with non-radioactive T4, thus reflecting a stronger affinity of this hormone for muscle deiodinating sites. This correlate with the greater displacment of [125I]T3 as revealed by the percentage of recovered labelled hormone. In 18 experiments liver homogenates deiodinated a mean 14.6% of a tracer amount of [125I]T4 and 8.5% of [125I]T3. The addition of a T4- or a T3-load was followed by a smaller decrease in percentual deiodination of both labelled hormones as compared to muscle homogenates. Unlike the effects observed in muscle, the breakdown of [125I]T4 and [125I]T3 by liver homogenates was equally affected by similar amounts of stable T4 or T3. It is concluded that in the present in vitro system T4 and T3 share cellular sites of deiodination in rat muscle and liver and that, at least in muscle, which constitutes over one-half of the rat body weight T4 appears to be preferentially deiodinated.     

Evaluation of two commercial kits for serum gastrin assay, and comparison with a conventional radioimmunoassay procedure

We compared two gastrin radioimmunoassay kits ("Immutope" kit, Squibb & Co.; "Gastrin R.I.A." kit, Schwarz/Mann) to the conventional gastrin radioimmunoassay of Yalow and Berson [Gastroenterology 58, 1 (1970)] as run by us and by a second reference laboratory. Although both kits were found to effectively discriminate above-normal and normal values for serum gastrin, they significantly underestimated very high values (greater than 1500 ng/liter). The Schwarz/Mann kit clearly had a superior quality label (lower nonspecific binding and higher specific activity) and a shorter incubation time. However, the 90-min incubation period cited for their kit caused overestimation of gastrin values in the lower range (5-300 ng/liter), which could be corrected by prolonging the incubation to 24 h. The Squibb antibody had fairly good cross reactivity to all gastrin species tested; the Schwarz/Mann antibody had poor affinity for natural human gastrin G34-II. Good correspondence was found for sera run by both reference laboratories (y = 0.96x + 10, r = 0.997), and values obtained with the Schwarz/Mann kit correlated best (+ 0.815) with those from the conventional radioimmunoassay procedure.     

Competitive binding assay for determination of rat insulin-like growth factor binding protein-3

We describe a novel competitive assay for rat insulin-like growth factor (IGF)-binding protein-3 (rIGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid labile subunit (ALS) in the presence of IGF. Human (h)ALS was bound to test tubes pre-coated with anti-human ALS antibody. The assay depends on competition between a covalent complex of 125I-hIGF-I and hIGFBP-3, added as tracer, and hIGFBP-3 or rIGFBP-3 in standards and test samples, for binding to the immobilized hALS. Purified natural hIGFBP-3 served as standard. Human IGFBP-3 and rIGFBP-3 were able to compete for tracer binding in the presence, but not in the absence, of IGF-I. Before assay, rat serum samples were acidified to denature endogenous ALS. Standards ranged from 0.10 (lower detection limit) to 20 ng/tube. Rat serum, semipurified rIGFBP-3, human serum and purified hIGFBP-3 diluted in parallel. The level of rIGFBP-3 was 1.63+/-0.28 mg/l (mean+/-SEM) in young rats and increased to 3.41+/-0.26 mg/l (p < 0.05) in old rats (n = 5-6). Fasting for 3 days reduced rIGFBP-3 from 2.41+/-0.27 to 1.33+/-0.14 mg/l (p < 0.05). Levels of rIGFBP-3 were reduced in hypophysectomized (0.16+/-0.04 mg/l; p < 0.05) and diabetic rats (1.04+/-0.30 mg/l; p < 0.05), and normal in insulin-treated diabetic rats (2.49+/-0.04 mg/l; ns), when compared to controls (2.79+/-0.22 mg/l). Changes in levels of IGFBP-3 parallelled those of immunoreactive rALS. We conclude that this assay provides a novel method of quantitating functional IGFBP-3 in rat serum.     

Triiodothyronine binding to lymphocytes from euthyroid subjects and a patient with peripheral resistance to thyroid hormone

Triiodothyronine (T3) binding to Ficoll-Isopaque purified human lymphocytes was studied. During incubation of lymphocytes with [125I]T3 in a calcium-free medium at 37 degrees C, maximal uptake of T3 in nuclei occurrred after 2 h and declined after prolonged incubationd incubation . Incubation of lymphocytes with T3 concentrations ranging from 1 X 10(-11) TO 1 X 10(-9) mol/l and subsequent treatment with Triton X-100 to strip off [125I]T3 bound with low affinity was used for the estimation of affinity and capacity of nuclear T3 binding sites. The mean equilibrium affinity constant (Ka) estimated with the Scatchard method in 11 euthyroid healthy subjects was 4.5 X 10(9) l/mol, and the mean maximal binding capacity 25 X 10(-5) mol/100 mug DNA. In a female patient with peripheral resistance to thyroid hormone action, the estimated Ka was 3.5 X 10(9) l/mol and the number of T3 binding sites 37 X 10(-15) mol/100 mug DNA. Although not statistically different from the mean value in euthyroid subjects, this Ka value was outside the range of control values observed and was considered presumptive evidence that the nuclear T3 receptors in this patient have abnormally low affinity for its ligand. The nuclear T3 binding capacity in this patient was significantly increased.