Adsorption of erythropoietin and growth hormone to peritoneal dialysis bags and tubing.

OBJECTIVE: To study the adsorption of erythropoietin and growth hormone to dialysis bags and tubing. DESIGN: In vitro study in which radiolabeled erythropoietin and recombinant human growth hormone were added to small-volume (50- and 250-mL) dialysis bags. Recovery was measured after 15-minute dwells. Experiments were performed in triplicate. SETTING: University hospital. RESULTS: Adsorption of erythropoietin and growth hormone was less than 7%. CONCLUSION: Adsorption of erythropoietin and recombinant human growth hormone to dialysis bags and tubing is minimal. This finding provides another argument in favor of intraperitoneal therapy in pediatric peritoneal dialysis.

Relationship between free and total 1,25-dihydroxyvitamin D in conditions of modified binding.

OBJECTIVE: A novel assay was employed to study the free 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations in various populations with different levels of 1,25(OH)2D and vitamin D binding protein (DBP). DESIGN: In 12 healthy women before and after 3 months of oral estrogen/progestagen treatment, 10 pregnant women, and 16 patients with a nephrotic syndrome and normal renal function, the concentrations of total and free 1,25(OH)2D, DBP and albumin were assessed. METHODS: The total concentration of 1,25(OH)2D in serum was assessed using a radioreceptor assay. The free fraction of 1,25(OH)2D was measured using symmetric dialysis. DBP was assessed using single radial immunodiffusion. Serum albumin concentrations were measured on an automated analyzer. RESULTS: In healthy women, the concentrations of total 1,25(OH)2D, free 1,25(OH)2D and DBP were 132+/-19 pmol/l, 92+/-30 fmol/l and 5.59+/-0.43 micromol/l. After 3 months of estrogen/progestagen treatment, total 1,25(OH)2D and DBP levels rose significantly to 175+/-51 pmol/l and 8.32+/-1.59 micromol/l (P< or =0.05 and P< or =0.001); the free 1,25(OH)2D remained unchanged (105+/-39 fmol/l; not significant). Pregnant women had significantly higher levels of total 1,25(OH)2D and DBP (239+/-68 pmol/l and 11.32+/-1.77 micromol/l; both P

Osteopenia in insulin-dependent diabetes mellitus; prevalence and aspects of pathophysiology.

The objective was to evaluate the prevalence and severity of osteopenia in patients with uncomplicated insulin-dependent diabetes mellitus (IDDM) and to obtain more information on the pathophysiology of diabetic osteopenia. In 35 patients with uncomplicated IDDM (21 men and 14 women; age 37.6+/-9.9 yr; duration of disease 8.5+/-3.5 years) bone mineral density was measured by dual energy X-ray absorptiometry (DEXA). In addition, markers of bone formation [plasma insulin-like growth factor I (IGF-I), serum alkaline phosphatase (ALP), serum bone alkaline phosphatase (BAP) and serum osteocalcin] and bone resorption [urinary excretion of calcium and of the cross-linked N-telopeptide of type 1 collagen, both corrected for the excretion of creatinine] were measured in the diabetic patients and in 33 healthy controls, matched for sex, age, height, weight and body mass index (BMI). In 67% of the diabetic men and 57% of the diabetic women osteopenia of the femoral neck and/or the lumbar spine (T-value < or = -1 SD) was present. Fourteen percent of the male patients, but none of the female patients, met the criteria for osteoporosis (T-value < or = -2.5 SD). In the whole group of diabetic patients the mean plasma IGF-I level tended to be lower (p<0.10) as compared to that in the controls. In the diabetic patients with femoral neck osteopenia, the mean plasma IGF-I level was significantly lower (p<0.05) than in those without osteopenia at this site. There were no differences in the mean serum ALP, BAP and osteocalcin levels between the diabetic patients and the controls, nor between the diabetic patients with and without femoral neck osteopenia. Considering only the male diabetic patients, significantly lower mean plasma IGF-I (-26%), serum ALP (-24%) and serum osteocalcin (-38%) levels were present in the patients with femoral neck osteopenia than in those without osteopenia at this site, suggesting lowered bone formation. The bone resorption markers were similar in all (sub)groups of diabetic patients and not different between diabetic patients and controls. Bone mineral density (BMD) did not correlate with plasma levels of glycosylated hemoglobin (HbA1c). BMD values were not related to any of the bone resorption or formation markers, except for plasma IGF-I both in the femoral neck (r=+0.38, p=0.026) and the lumbar spine (r=+0.34, p=0.043). Our data demonstrate that at least in male patients with IDDM, osteopenia is the consequence of a lowered bone formation with a predominance of bone resorption over formation.

Female sex hormone replacement therapy increases serum free 1,25-dihydroxyvitamin D3: a 1-year prospective study.

OBJECTIVE: Currently, hormone replacement therapy is applied successfully to reduce post-menopausal bone resorption. However, the exact mechanism by which oestrogen exerts its effect has not yet been fully elucidated. In order to determine whether changes in the biologically active 1,25-dihydroxyvitamin D3 may be of importance in this process, the concentrations of both total and free 1,25-dihydroxyvitamin D3 in serum were assessed. DESIGN: In 36 post-menopausal women the effect of hormone replacement therapy, with a combination of 17 beta-oestradiol and norethisterone acetate, on the serum levels of total and free 1,25-dihydroxyvitamin D3 was studied after 0, 3, 6 and 12 cycles. MEASUREMENTS: The total concentration of 1,25-dihydroxyvitamin D3 in serum was assessed using a radioreceptor assay after diethylether extraction of the samples followed by paper chromatography. The free fraction of 1,25-dihydroxyvitamin D3 was measured using symmetric dialysis. The free 1,25 dihydroxyvitamin D3 concentration was calculated by multiplying the total concentration by the free fraction. RESULTS: During therapy, mean serum total 1,25-dihydroxyvitamin D3 concentrations (+/- SD) were 106.4 pmol/l (+/- 27.5), 155.0 pmol/l (+/- 49.5), 176.7 pmol/l (+/- 70.0) and 161.1 pmol/l (+/- 55.3) at 0, 3, 6 and 12 cycles, respectively. Serum free 1,25-dihydroxyvitamin D3 concentrations were 68 fmol/l (+/- 22), 107 fmol/l (+/- 35), 120 fmol/l (+/- 43) and 108 fmol/l (+/- 37), respectively. Baseline values of both total and free 1,25-dihydroxyvitamin D3 were significantly lower than those during therapy at all time (P < or = 0.001). CONCLUSION: Both the serum total 1,25-dihydroxyvitamin D3 and the serum free 1,25-dihydroxyvitamin D3 concentrations are increased during combined 17 beta-oestradiol and norethisterone acetate therapy for a year. Assuming that the free concentration 1,25-dihydroxyvitamin D3 reflects the biologically active fraction, this rise may in part explain the preventive effect of hormone replacement therapy on osteoporosis.

Immune responses to experimental stress: effects of mental effort and uncontrollability.

OBJECTIVE: Two important determinants of physiological stress responses have been identified, uncontrollability of the stressor and amount of effort involved in coping with the stressor. In the present experiment, we tried to identify the specific contributions of effort and uncontrollability to immune system responses to stress. METHODS: In a 2 x 2 design, effort and uncontrollability were manipulated independently of each other. Subjects participated in one of four experimental conditions, and their endocrine, immune, and sympathetic nervous system responses to the task were assessed. RESULTS: Effort had a stimulating effect on enumerative immunological parameters (CD8 and CD16+ cells) and on natural killer cell activity. The effect occurred immediately after the stressor and was transient. Regression models indicated that this effort effect may have been mediated by activation of the sympathetic nervous system. Uncontrollability influenced in vitro production of the cytokine interleukin-6, leading to decreased production 15 and 30 minutes after the stressor. Uncontrollability also led to an increased level of cortisol, but no evidence was found that the decrease in cytokine production was mediated by cortisol release. CONCLUSION: The results suggest that two major stressor characteristics, effort and uncontrollability, may have differential effects on the immune system.

Plasma IGF-I is a useful marker of growth hormone deficiency in adults.

Diagnosing growth hormone deficiency in adults is difficult. Provocation tests are most commonly used for the diagnosis with the insulin-induced hypoglycemia test nowadays considered as the "gold standard". The role of IGF-I concentrations in diagnosing growth hormone deficiency in adults is under discussion. In 58 adult patients with proven growth hormone deficiency, the sensitivity and specificity of IGF-I concentrations in relation to growth hormone deficiency were evaluated. Reference values of plasma IGF-I were obtained from 53 healthy volunteers. Using a calculated cut-off concentration of 15 nmol/l we were able to demonstrate that IGF-I concentration is a reliable screening method for growth hormone deficiency. Using this cut-off point in a patient population younger than 40 years of age, sensitivity was 90% and specificity 89%. For patients exceeding the age of 40 years, sensitivity, specificity and positive predictive value were rather low, but the negative predictive value was as high as 90%, indicating that for patients over 40 years IGF-I concentrations above 15 nmol/l exclude growth hormone deficiency. In summary, under the age of 40 years measuring plasma IGF-I provides an useful tool to diagnose growth hormone deficiency, whereas above 40 plasma IGF-I values exceeding 15 nmol/l virtually exclude growth hormone deficiency.

Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by symmetric (rate) dialysis.

Most of the total circulating 1,25-dihydroxyvitamin D (1,25(OH)2D) is bound to plasma proteins, mainly vitamin D-binding protein (DBP) and albumin. Only a small fraction in plasma exists in the free form. It is widely assumed that the non-protein-bound free hormone reflects the biologically active fraction. We describe a dialysis method for the determination of plasma free 1,25(OH)2D which is relatively easy to perform. In this symmetric or "rate" dialysis method, identical samples are placed at both sides of a membrane. At one side, tritiated 1,25(OH)2D is added and the rate of transfer of this tritiated 1,25(OH)2D through a dialysis membrane is directly related to the free fraction of plasma 1,25(OH)2D. This method is much less susceptible toward tracer impurities than indirect equilibrium dialysis and centrifugal ultrafiltration. Moreover, it requires much less tracer. The intraassay coefficient of variation for the determination of the free fraction is 1.0%; the interassay variation is 7.7%. Comparison of the free fraction of 23 samples assessed with both centrifugal ultrafiltration and symmetric dialysis showed much higher values using the former method. No significant correlation between the two methods was found. The free fraction of 1,25(OH)2D in normal subjects as assessed with symmetric dialysis ranges from 0.049 to 0.103%.

[Hypertension caused by licorice consumption]. / Hypertensie door dropgebruik.

In a 38-year-old woman who was hospitalized because of hypertension and hypokalaemic alkalosis, the intake of liquorice (200 g per day) was proven to be the cause. A liquorice provocation test produced all the expected clinical and biochemical abnormalities. Some kinds of liquorice contain glycyrrhetic acid which inhibits the enzyme 11-beta-hydroxysteroid dehydrogenase (e.g. in the kidney) leading to decreased transformation of cortisol into cortisone. The mineralocorticoid action of cortisol causes a drop in serum potassium and an increase in serum sodium concentration, together with a metabolic alkalosis, which in the patient described led to retention of water resulting in weight increase and hypertension.

Comparison of the antileukemic activity in vitro of dexamethasone and prednisolone in childhood acute lymphoblastic leukemia.

It is generally assumed that prednisolone (PRD) and dexamethasone (DXM) have equal glucocorticoid activity of PRD is given at sevenfold higher doses. Results of clinical studies of childhood acute lymphoblastic leukemia (ALL) suggested that DXM is more potent relative to PRD than assumed. The purpose of this study was to determine the relative antileukemic activity of PRD phosphate and DXM phosphate in 133 untreated childhood ALL samples in vitro, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. There was a marked variation in antileukemic activity of both agents among the patient samples. The median LC50 (drug concentration lethal to 50% of the ALL cells) for PRD phosphate was 3.50 microM, for DXM phosphate 0.20 microM. The individually calculated ratios of the LC50 values for PRD and DXM phosphate showed a large range from 0.7 to >500, with a median of 16.2. This 16-fold difference could not be explained by differences between these glucocorticoids in stability, hydrolysis into unesterified drug, adhesion to the wall of the microculture plates, or protein binding. ALL cells were cross-resistant to PRD and DXM phosphate (correlation coefficient = 0.85, P<0.000001). We conclude that the in vitro antileukemic activity of DXM phosphate is median 16-fold higher than that of PRD phosphate, which contrasts to the generally assumed factor of 7. Based on the higher potency of DXM, and its more favorable pharmacokinetics as reported in the literature, DXM may be preferred to PRD as the glucocorticoid in the treatment of ALL.

Fluticasone propionate does not influence bone metabolism in contrast to beclomethasone dipropionate.

Inhaled corticosteroids (ICS) in dosages above 1,000 micrograms/d may influence parameters of bone metabolism. Fluticasone propionate (FP) is a new ICS with a higher clinical potency than beclomethasone dipropionate (BDP) combined with negligible oral bioavailability. The aim of this study was to evaluate the effects of FP and BDP in clinically equipotent dosages on indices of bone metabolism and morning cortisol. FP 750 micrograms/d and BDP 1,500 micrograms/d were compared in a randomized, double-blind, crossover study consisting of two 6-wk treatment periods, each preceded by a 3-wk single-blind placebo period. Twenty-one nonsmoking asthmatic completed the study. FP had the same effect on FEV1 and peak expiratory flow as the double dose of BDP. Both treatments did not change morning cortisol. BDP decreased both osteocalcin and procollagen type 1 carboxyterminal propeptide, indices of bone formation, significantly by 18.5 and 21.9%, respectively. In contrast, FP did not change any variable of bone formation. FP and BDP did not increase type 1 collagen carboxyterminal telopeptide and deoxypyridinoline crosslinks, both markers of bone resorption. In changes in parameters of bone metabolism indicate adverse effects on bone quality in the long term, FP may offer an advantage over BDP.

Thyroid function studies in normal pregnant Tanzanian women.

Iodine deficiency is well known as a cause of several disorders such as endemic goiter and cretinism, along with a wide spectrum of psychoneurologic development disorders including endemic mental deficiency, which are generally correlated with damage to the fetus. Since as much as 40% of the Tanzanian population is at risk for iodine deficiency disorders (IDD) because they live in iodine-deficient areas, and although the effects of iodine deficiency on human reproduction in Tanzania have not been objectively studied, it is estimated that there are approximately 600,000 cretins and cretinoids in the country as a result of IDD. As a baseline study for future research on iodine deficiency and its effects on human reproduction in Tanzania, we assayed serum thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), and free thyroxine (FT4) in 93 clinically euthyroid pregnant women and 34 nonpregnant women as controls. Pregnancy was accompanied by significantly increased levels of total T3 and T4, decreased FT4, and increased TSH concentration in serum. However, biochemical euthyroidism (assessed by FT4 and basal TSH) was demonstrated in almost all (99%) of the pregnant subjects in conformity with most of the previous findings elsewhere. We conclude that pregnant Tanzanian women residing in areas without iodine deficiency experience changes in biochemical parameters of thyroid function similar to their counterparts in other places.

Reliability of salivary testosterone measurements: a multicenter evaluation.

The reliability of salivary testosterone assays was evaluated by nine laboratories in four countries. Each laboratory used its own RIA procedures to assay samples from a set of 100 male and 100 female subjects. Agreement among the laboratories on mean scores was within the range reported by Read (Ann N Y Acad Sci 1993; 694: 161-76). Overall agreement on individual scores, as indicated by the intraclass correlation coefficient computed within subjects across laboratories, was r = 0.87 for men and r = 0.78 for women. Mean agreement between each laboratory and the combined set of all other laboratories (via Fisher's Z-transformation) was r = 0.61 for men and r = 0.58 for women. We take these latter values to be the best estimates of the average reliability of laboratories in their ordering of individual samples.

Bone mineral density and bone turnover before and after surgical cure of Cushing's syndrome.

We measured bone mineral density (BMD) using dual-energy x-ray absorptiometry in 20 patients with Cushing's syndrome (CS) (14 pre- and 2 postmenopausal women, 4 men) before and in 18 of them also at regular intervals after surgical cure (median duration of follow-up, 36 months). In addition, in the premenopausal women with CS, fasting blood samples and 2-h fasting urine samples for measurement of biochemical parameters of bone and collagen metabolism were collected before and in 9 of them also at regular intervals during the first 2 yr after surgery. Marked osteopenia was present in most patients with active CS (Z-scores: lumbar spine -1.45 +/- 1.44 and femoral neck -1.50 +/- 1.02; mean +/- SD). No consistent change in BMD was observed at 3 and 6 months after surgery. Thereafter BMD increased considerably in almost all patients. For the 15 patients with a follow-up of at least 1 yr, Z-scores at the last evaluation were -0.65 +/- 1.27 for the lumbar spine and -0.98 +/- 1.02 for the femoral neck (both P < 0.002 compared with pretreatment values). In the premenopausal patients, the increase in BMD both in the lumbar spine and in the femoral neck at 24 months was inversely correlated with age (r = -0.733, P < 0.03, and r = -0.667, P < 0.05, respectively). Serum levels of osteocalcin, bone alkaline phosphatase, carboxyterminal propeptide of type I procollagen, aminoterminal propeptide of type III procollagen, and the cross-linked telopeptide of type I collagen were not significantly different between the group of 14 premenopausal patients with active CS and a control group of 18 age-matched healthy premenopausal women. However, the urinary hydroxyproline/creatinine ratio was significantly higher in patients with CS (24.6 +/- 9.6 vs. 16.2 +/- 3.5 mumol/mmol, P < 0.01). In all 9 premenopausal patients, serum levels of osteocalcin increased considerably between 0 and 3 months (from 1.04 +/- 0.20 to 3.82 +/- 0.30 nmol/L) (mean +/- SEM, P < 0.0001), indicating a prompt increase of osteoblast activity. Also serum levels of carboxyterminal propeptide of type I procollagen, aminoterminal propeptide of type III procollagen, and cross-linked telopeptide of type I collagen, and the urinary hydroxyproline/creatinine ratio increased significantly between 0 and 3 months. Thereafter these levels decreased gradually. We conclude that marked osteopenia in the lumbar spine and femoral neck is present in most patients with active Cushing's syndrome.(ABSTRACT TRUNCATED AT 400 WORDS)

Highly sensitive and specific HPLC with fluorometric detection for determination of plasma epinephrine and norepinephrine applied to kinetic studies in humans.

An HPLC separation method combined with fluorometric detection was extended to enable simultaneous assessment of plasma 3H-labeled and endogenous epinephrine (E) and norepinephrine (NE). Forearm fractional extraction (FFE) of 3H-labeled E and NE and of endogenous E was measured in 40 healthy volunteers who were receiving a continuous infusion of 3H-labeled E and NE. Concentrations of arterial and venous E were 26.8 +/- 1.95 (mean +/- SE) and 6.8 +/- 0.75 ng/L, respectively. Arterial and venous NE and dopamine (DA) were also measured, with respective values of 140.7 +/- 8.5 and 192.1 +/- 15.1 for NE, and 13.1 +/- 0.78 and 11.3 +/- 0.70 ng/L for DA. The FFE of 3H-labeled E was slightly but significantly higher (0.790 +/- 0.016) than the that of either 3H-labeled NE or endogenous E (0.748 +/- 0.0146 and 0.745 +/- 0.0185, respectively; P < 0.001), the correlations being highly significant (r = 0.80, P < 0.001) in both cases. The small difference between the FFE of E and of 3H-labeled E allows the calculation of the apparent spillover of E. However, this spillover was negligible compared with forearm NE spillover (0.0112 +/- 0.0031 vs 1.369 +/- 0.128 ng/L per minute. The high sensitivity of this measurement of venous E widens the possibilities for studying E kinetics under physiological conditions.

Hormone replacement therapy increases serum 1,25-dihydroxyvitamin D: A 2-year prospective study.

Osteoporosis is a common disorder in postmenopausal women, which is probably due to decreased ovarian function. Currently, hormone replacement therapy (HRT), involving administration of estrogen and progestogen, is successfully applied to reduce bone resorption. We studied the effect of HRT on 23 postmenopausal women. This consisted of a combination of 17 beta-estradiol and dydrogesterone, on the serum level of 1,25-dihydroxyvitamin D (1,25(OH)2D) after 0, 6, 12, and 24 months. We found mean serum concentrations (+/- SD) of 1,25(OH)2D of 130.5 pmol/liter (46.1), 152.7 pmol/liter (45.1), 170.8 pmol/liter (64.0), and 155.2 pmol/liter (59.7), respectively. The baseline values in these women were found to be significantly lower than those during therapy (P < or = 0.005). No statistically significant differences were observed when comparing the estrogen-only phase with the combined estrogen-progestogen phase. It is concluded that HRT results in an increase in the serum 1,25(OH)2D concentration which lasts for at least 2 years. This increase may partly explain the preventive effect of HRT on osteoporosis. Furthermore, these results suggest that dydrogesterone does not influence the estrogen-induced changes in serum 1,25(OH)2D concentration.

Advantages of paper chromatography as a preparative step in the assay of 1,25-dihydroxyvitamin D.

Ether extraction and paper chromatography were used to separate the main metabolites of vitamin D in plasma [25-(OH), 24,25-(OH)2 and 1,25-(OH)2 vitamin D]prior to radio receptor-assay. The overall procedural loss of the 1,25-(OH)2 vitamin D was 58 +/- 5% (n = 40), corrected for by tracer addition. The sensitivity of the assay was 0.5 fmol/tube, corresponding to 4 pmol/l, and the intra- and inter-assay coefficients of variation were 10.5% and 11.5%, respectively. The range of values measured in healthy controls was 80-200 pmol/l (n = 60), which is in agreement with findings reported in the literature. A comparison of the results of the present procedure with those obtained with a procedure employing C18 purification, disclosed a correlation coefficient of 0.92 (p < or = 0.0001), a slope of 0.89 (p < or = 0.0001) and a small non-significant intercept of 5.0 pmol/l (n = 53).

Low ratio of androstenedione to testosterone in plasma and saliva of hirsute women.

To gain insight into the passage of androstenedione (A4) through the salivary gland, we measured concentrations of plasma total (TA4), free (FA4), and salivary (SA4) androstenedione during administration of dexamethasone and synthetic corticotropin to control subjects and hirsute women and compared these data with plasma total (TT), free (FT), and salivary testosterone (ST) concentrations. TA4 and FA4 were significantly lower in control subjects throughout (0, 15, 45, and 75 min) (P less than 0.05). SA4 in control subjects was significantly lower only in the follicular phase at 0 and 15 min. There was no significant difference between the increments in SA4 in response to corticotropin. The concentration of SA4 in the control women at 0 min was not significantly different from the concentration of FA4. At 45 and 75 min after corticotropin administration, however, SA4 was slightly higher than FA4 (P less than 0.01). In hirsute women, however, the concentration of SA4 was significantly lower than FA4 at all times (P less than 0.05). TT, FT, and ST concentrations were about twofold higher in the hirsute women than in control subjects throughout. In both groups, ST concentrations were three times as high as FT concentrations (P less than 0.001). The SA4:ST ratio was significantly lower than the FA4:FT ratio in both groups (P less than 0.001) because of higher ST than FT concentrations and similar or even lower SA4 concentrations in both groups. Both FA4:FT and SA4:ST ratios were lower in hirsute women, except for the FA4:FT ratio in control subjects in the luteal phase. Our data are compatible with 17-hydroxysteroid oxidoreductase activity in the salivary gland. If the difference in the ratios of A4 to T between hirsute women and control subjects is attributed to this hypothetical enzymatic activity, it would suggest a more rapid conversion of A4 to T in the hirsute group.

Scintillation proximity assay to study the interaction of epidermal growth factor with its receptor.

Scintillation Proximity Assay (SPA), which does not require the physical separation of receptor bound and free ligand, was applied to study the interaction of Epidermal Growth Factor (EGF) with its receptor (EGFR) in membrane preparations from human placenta. Fluomicrospheres to which the monoclonal anti-EGFR antibody R1 was coupled, were used. Kinetic binding data of the association of 125I-labeled EGF binding to the receptor at 20 degrees C could be fitted according to a double exponential model, which is consistent with the presence of fast and slow associating EGF binding sites. Dissociation kinetics revealed that perturbation of equilibrium conditions rapidly occurs upon washing. Multiple point Scatchard analysis of equilibrium 125I-labeled EGF binding data revealed curvilinearity, indicating the presence of both high and low affinity EGF binding sites. We conclude that SPA is an interesting new tool in the exploration of the interaction of ligands with their receptors, which allows detailed ligand-receptor studies under precise in situ conditions.

Six methods for direct radioiodination of mouse epidermal growth factor compared: effect of nonequivalence in binding behavior between labeled and unlabeled ligand.

Mouse epidermal growth factor (EGF) was radioiodinated by six different direct iodination methods. The 125I-labeled EGF preparations were distinguished by analyzing the binding of the radioligand to the EGF receptor (EGFR)-containing human placental membranes. The receptor-binding affinity of EGF labeled with Chloramine T was less than the affinity of unlabeled EGF, which precluded an accurate determination of the specific radioactivity of the 125I-labeled EGF preparation by "self-displacement analysis." Scatchard analysis of competitive binding data (increasing concentrations of unlabeled EGF) obtained with commercially prepared 125I-labeled EGF (Chloramine T method), according to the specific radioactivity stated by the manufacturer, resulted in a substantial underestimation of the apparent number of receptors. Iodination of EGF with Iodogen or Iodo-beads, reagents claimed to be more gentle because of their solid state, also yielded 125I-labeled EGF preparations that were not equivalent to the native EGF in receptor binding. In contrast, equivalence in the ligand-receptor interaction between labeled and unlabeled EGF could be achieved by iodinating EGF with iodine monochloride (ICl), Protag-125, or lactoperoxidase-glucose oxidase-coupled beads (Enzymobeads). Scatchard plots of saturation and competitive binding data obtained with these 125I-labeled EGF preparations produced identical results for apparent receptor number and apparent dissociation constants. Such radioiodinated EGF preparations yield relevant binding data in competition studies of labeled and unlabeled EGF.