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Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST -associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

Background and Purpose: Recent advances in molecular techniques have characterized distinct subtypes of diffuse intrinsic pontine gliomas. Our aim was the identification of MR imaging correlates of these subtypes., Materials and Methods: Initial MRIs from subjects with diffuse intrinsic pontine gliomas recruited for a prospective clinical trial before treatment were analyzed. Retrospective imaging analyses included FLAIR/T2 tumor volume, tumor volume enhancing, the presence of cyst and/or necrosis, median, mean, mode, skewness, kurtosis of ADC tumor volume based on FLAIR, and enhancement at baseline. Molecular subgroups based on EGFR and MGMT mutations were established. Histone mutations were also determined ( H3F3A, HIST1H3B, HIST1H3C ). Univariate Cox proportional hazards regression was used to test the association of imaging predictors with overall and progression-free survival. Wilcoxon rank sum, Kruskal-Wallis, and Fisher exact tests were used to compare imaging measures among groups., Results: Fifty patients had biopsy and MR imaging. The median age at trial registration was 6 years (range, 3.3-17.5 years); 52% were female. On the basis of immunohistochemical results, 48 patients were assigned to 1 of 4 subgroups: 28 in MGMT -/ epidermal growth factor receptor ( EGFR )-, 14 in MGM T-/EGFR+, 3 in MGMT +/EGFR-, and 3 in MGMT +/EGFR+. Twenty-three patients had histone mutations in H3F3A, 8 in HIST1H3B, and 3 in HIST1H3C. Enhancing tumor volume was near-significantly different across molecular subgroups ( P  = .04), after accounting for the false discovery rate. Tumor volume enhancing, median, mode, skewness, and kurtosis ADC T2-FLAIR/T2 were significantly different ( P  ≤ .048) between patients with H3F3A and HIST1H3B/C mutations., Conclusions: MR imaging features including enhancement and ADC histogram parameters are correlated with molecular subgroups and mutations in children with diffuse intrinsic pontine gliomas., (© 2020 by American Journal of Neuroradiology.)

The success of locomotion training with robotic exoskeletons requires identifying control algorithms that effectively retrain gait patterns in neurologically impaired individuals. Here we report how the two training paradigms, performance-based error-augmentation versus error-reduction, modified walking patterns in four chronic post-stroke individuals as a proof-of-concept for future locomotion training following stroke. Stroke subjects were instructed to match a prescribed walking pattern template derived from neurologically intact individuals. Target templates based on the spatial paths of lateral ankle malleolus positions during walking were created for each subject. Robotic forces were applied that either decreased (error-reduction) or increased (error-augmentation) the deviation between subjects' instantaneous malleolus positions and their target template. Subjects' performance was quantified by the amount of deviation between their actual and target malleolus paths. After the error-reduction training, S1 showed a malleolus path with reduced deviation from the target template by 16%. In contrast, S4 had a malleolus path further away from the template with increased deviation by 12%. After the error-augmentation training, S2 had a malleolus path greatly approximating the template with reduced deviation by 58% whereas S3 walked with higher steps than his baseline with increased deviation by 37%. These findings suggest that an error-reduction force field has minimal effects on modifying subject's gait patterns whereas an error-augmentation force field may promote a malleolus path either approximating or exceeding the target walking template. Future investigation will need to evaluate the long-term training effects on over-ground walking and functional capacity.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to measure 6 metabolic compounds of the adrenocorticosteroid pathway simultaneously on residual specimens from patients who had previously been previously diagnosed, on the basis of immunoassays, as having congenital adrenal hyperplasia (CAH), 11 beta-hydroxylase deficiency, 21-hydroxylase deficiency, or Addison disease (adrenal insufficiency). Two subjects with normal adrenal function had serum cortisol values of 13.6 and 8.9 micrograms/dL and serum cortisone values of 2.1 and 0.6 microgram/dL, but the rest of the compounds were undetectable. Two patients with 11 beta-hydroxylase deficiency had serum 11 beta-deoxycortisol values of 14.9 and 10.0 micrograms/dL and serum 11-deoxycorticosterone values of 3.9 and 1.0 microgram/dL, but their serum levels of cortisol and cortisone were diminished. A patient with 21-hydroxylase deficiency had a highly increased serum 17-hydroxyprogesterone concentration of 28.5 micrograms/dL (or 28,500 ng/dL, the traditional unit to report this assay) and a serum 21-deoxycortisol concentration of 6.9 ug/dL (this is a pathologic marker of 21-hydroxylase deficiency that is nondetectable in sera of healthy subjects). This patient also had diminished concentrations of serum cortisol and cortisone (0.9 and 0.3 microgram/dL, respectively). At 30 and 60 min after corticotropin (ACTH) stimulation, serum cortisol was the only compound that showed a dramatic increase in the normal subjects; the patient with 21-hydroxylase deficiency showed an increase of serum 17-hydroxyprogesterone level, but no increase of serum cortisol level; the patient with Addison disease showed no increase in the levels of serum cortisol or other compounds. Metyprapone, which blocks 11 beta-hydroxylase activity, increased the serum 11-deoxycorticosteroid levels and decreased the serum cortisol level. This pilot study demonstrates that it is feasible to use LC-MS/MS for the laboratory diagnosis of adrenal cortical dysfunction. The authors envision that LC-MS/MS may soon become an ideal analytical technique for the diagnosis of such endocrine diseases.

Current trends in the management of type 2 diabetes mellitus, based on the 20-year United Kingdom Prospective Diabetic Study, include intensive treatment to control the blood glucose level and blood pressure in order to prevent or delay microvascular and cardiovascular complications. In the new millennium, type 2 diabetes will become epidemic in developing countries. If diabetes were to develop in 10% of the 1.2 billion population of China, the expense of intensive treatment would be immense. Laboratory tests are useful for detecting risk factors before the onset of the disease and convincing the general public to take preventive measures. Glucose tolerance testing is one of these tests. When glucose tolerance is impaired, 25% of beta-cell function is lost. Determining the plasma proinsulin level is another useful evaluation; impaired glucose tolerance accompanied by increased plasma proinsulin level is indicative of an enhanced risk that type 2 diabetes will develop within 5 years. Educating the public about eating a healthy diet and exercising may prevent the development of diabetes and thereby reduce the global prevalence of type 2 diabetes.

Fly ash from coal-fired thermal power plants can be used for the removal of 2-chlorophenol (2-CP) and 2,4-dichlorophenol (2,4-DCP) with enthalpy changes of about -3 kcal/mol. The amounts of 2-CP and 2,4-DCP removed are affected by the pH value of the solution. The efficiency of removal improves when the pH value is less than the pK(a) values of 2-CP and 2,4-DCP, respectively. The adsorbed amount of chlorophenol by fly ash is also affected by particle diameter, carbon content, and the specific surface area of the ash used in this study. As expected, more adsorption takes place with fly ash of higher carbon content and larger specific surface area. Moreover, the adsorbed amount of chlorophenol is not influenced by the matrix in the wastewater, as shown by studying the removal of 2-CP and 2, 4-DCP in wastewater from a synthetic fiber plant. Chlorophenols in the wastewater were also removed efficiently through a fly ash column, with breakthrough times being inversely proportional to flow rates.

Impaired insulin secretion in type 2 diabetes is characterized by decreased first-phase insulin secretion, an increased proinsulin-to-insulin molar ratio in plasma, abnormal pulsatile insulin release, and heightened disorderliness of insulin concentration profiles. In the present study, we tested the hypothesis that these abnormalities are at least partly reversed by a period of overnight suspension of beta-cell secretory activity achieved by somatostatin infusion. Eleven patients with type 2 diabetes were studied twice after a randomly ordered overnight infusion of either somatostatin or saline with the plasma glucose concentration clamped at approximately 8 mmol/l. Controls were studied twice after overnight saline infusions and then at a plasma glucose concentration of either 4 or 8 mmol/l. We report that in patients with type 2 diabetes, 1) as in nondiabetic humans, insulin is secreted in discrete insulin secretory bursts; 2) the frequency of pulsatile insulin secretion is normal; 3) the insulin pulse mass is diminished, leading to decreased insulin secretion, but this defect can be overcome acutely by beta-cell rest with somatostatin; 4) the reported loss of orderliness of insulin secretion, attenuated first-phase insulin secretion, and elevated proinsulin-to-insulin molar ratio also respond favorably to overnight inhibition by somatostatin. The results of these clinical experiments suggest the conclusion that multiple parameters of abnormal insulin secretion in patients with type 2 diabetes mechanistically reflect cellular depletion of immediately secretable insulin that can be overcome by beta-cell rest.

Two prospectively studied patients with polycythemia vera (PV) whose platelet counts showed marked periodic fluctuation during treatment with hydroxyurea (HU) are reported. Cycle lengths in both were approximately 28 to 30 days. In one patient, the cyclic process was no longer evident when treatment with HU was withheld, and it reappeared on treatment rechallenge. Circulating thrombopoietin (TPO) levels fluctuated out of phase with the platelet count despite markedly reduced TPO-receptor (c-Mpl) expression in bone marrow megakaryocytes. These observations suggest that the cyclic phenomenon may be related to both a transient state of HU-induced depletion of megakaryocytes and a concentration-dependent mitigation by TPO of the HU effect on megakaryocytes and their precursors. It is conceivable that the affected patients harbor a megakaryocyte progenitor pool whose apoptotic activity is differently modulated by either HU or high concentrations of TPO. (Blood. 2000;96:1582-1584)

Zinc modulates the structure and binding of the DNA binding domain of the 1alpha,25-dihydroxyvitamin D(3) receptor to specific vitamin D response element DNA (Nature Biotechnology 16, 262-266, 1998). To determine whether zinc alters 1alpha,25-dihydroxyvitamin D(3)-regulated genes in cells, we permanently transfected rat osteoblasts with two vitamin D-dependent promoter-reporter systems and examined their responses to 1alpha,25-dihydroxyvitamin D(3) in the presence of increasing amounts of extracellular zinc. When extracellular zinc concentrations were increased in the presence of 1alpha,25-dihydroxyvitamin D(3), there was an increase in the activity of 1alpha,25-dihydroxyvitamin D(3)-dependent promoters with increasing concentrations of zinc. The effect was specific for zinc since metals such as copper failed to increase the activity of 1alpha,25-dihydroxyvitamin D(3)-dependent promoters. The concentration of the vitamin D receptor within the cell and the affinity of 1alpha,25-dihydroxyvitamin D(3) for its receptor remained unchanged with added zinc. Our results show that zinc increases the activity of 1alpha,25-dihydroxyvitamin D(3)-dependent promoters in osteoblasts., (Copyright 2000 Academic Press.)

Abnormalities of carbohydrate metabolism in hyperthyroid patients have been long noted. Elevated proinsulin level is considered as an early marker of B-cell impairment. Proinsulin levels in hyperthyroid patients decreased after antithyroid drug therapy. However, proinsulin in hypothyroid patients was only rarely reported, and the difference was only demonstrated after glucose stimulation-there was a greater response of proinsulin secretion after thyroxine therapy-and the basal fasting proinsulin level was not different after therapy. One of the reasons might be that the assay was not sensitive enough to detect the change of basal proinsulin levels in patients with hypothyroidism after therapy. A newly developed immunochemiluminometric assay of proinsulin was used to demonstrate that the suppressed proinsulin level increased after thyroxine therapy in hypothyroid patients (4.2 +/- 2.4 vs. 10.0 +/- 5.6 pmol/L, p < 0.05; n = 7). On the other hand, our study also confirmed that the proinsulin levels decreased in hyperthyroid patients after antithyroid therapy by methimazole (27.8 +/- 26.0 vs. 15.8 +/- 15.7 pmol/L, p < 0.05; n = 12). In conclusion, proinsulin increased in hypothyroid patients after thyroxine therapy and decreased in hyperthyroid patients after methimazole therapy. The results demonstrated there is a high correlation between thyroid function and B-cell function in hypothyroid as well as hyperthyroid patients.

Objective: To report the high prevalence of increased parathyroid hormone-related peptide (PTHrP) in patients with islet cell carcinoma and associated hypercalcemia., Design: We conducted a retrospective study of PTHrP levels in patients with hypercalcemia and eucalcemia associated with islet cell carcinoma and compared these findings with those in healthy subjects., Material and Methods: Using a sensitive PTHrP immunochemiluminometric assay, we measured PTHrP levels in 17 patients with islet cell carcinoma and 110 healthy subjects. The differences between PTHrP levels in patients with normal and those with high serum calcium concentrations were analyzed statistically., Results: PTHrP levels were significantly higher (P < 0.01) in 10 patients with hypercalcemia and islet cell carcinoma (median, 14.0 pmol/L; range, undetectable to 40.1) than in 7 patients with eucalcemia and islet cell carcinoma (median, undetectable; range, undetectable to 1.3 pmol/L) or in the 110 healthy subjects (median, undetectable; range, undetectable to 4.2 pmol/L). The range of increased PTHrP levels in hypercalcemic islet cell carcinoma was 2 to 20 times the upper normal limit (2.0 pmol/L). Decreased PTHrP and serum calcium and increased parathyroid hormone levels were demonstrated in two patients after effective therapy. For all seven eucalcemic patients with islet cell carcinoma, PTHrP levels did not differ significantly from those in healthy subjects., Conclusion: PTHrP levels are increased in a substantial proportion of patients with hypercalcemia and islet cell carcinoma and seem to decrease after treatment of the underlying tumor. Measurement of PTHrP levels may be useful for confirming the diagnosis of hypercalcemia associated with malignant disease and for monitoring of therapy.

An immunochemiluminometric assay of parathyroid-hormone-related peptide (PTHrP) was developed with purified antisera produced from a single goat immunized with (1-86) PTHrP. One batch of purified antibodies was labeled with acridinium ester used as tracer antibodies; a second batch of the purified antibodies was immobilized onto plastic bead. Sensitivity of the assay was 0.1 pmol/L. The assay had no cross-reactivity with PTH. Seventy-five percent of healthy individuals had undetectable PTHrP (mean +/- S.D. were 0.73 pmol/L +/- 0.6; n = 110). The 95 percent upper reference limit was 2.0 pmol/L. Five of the seven patients with humoral hypercalcemia associated with malignancy (HHM) of solid-tumor had PTHrP greater than 2.0 pmol/L. The test efficacy in detecting HHM was 71 percent, which is similar to previous extraction radioimmunoassay (RIA) or immunoradiometric assay (IRMA). The new assay lowered the detection limit to 0.1 pmol/L, which is a great improvement from that of RIA at 2.0 pmol/L and IRMA at 1.0 pmol/L; however; it did-not improve the test efficacy in detecting patients with HHM. It may indicate the patients with HHM only have elevated plasma PTHrP in 70 percent of them. Technically, the method can be used as a model to develop chemiluminescent esoteric test for research or reference laboratories.

To determine the efficacy of cortisol and its metabolite, cortisone, measured simultaneously by high performance liquid chromatography (HPLC) in the diagnosis of Cushing's syndrome, we retrospectively reviewed the histories of 29 surgically proven Cushing's syndrome patients (20 Cushing's disease, 5 ectopic ACTH syndrome, and 4 adrenal Cushing's syndrome) and 6 patients with exogenous Cushing's syndrome. These 35 patients had urinary free cortisol determined by both HPLC and competitive binding methods. The efficacy of the HPLC assay using cortisol alone was equivalent to that of the competitive binding assay; 22 of 29 (76%) patients had increased cortisol. Cortisone also aided in the diagnosis; 25 of 29 (86%) had increased cortisone. Twenty-seven of the 29 (93%) patients had either both cortisone and cortisol (n = 19) or at least 1 of the 2 (n = 8) increased. All 6 patients with exogenous Cushing's syndrome had suppressed urinary free cortisol, cortisone, and the presence of prednisone and prednisolone. In the competitive binding assay, all exogenous Cushing's patients had falsely increased cortisol results. In conclusion, urinary free cortisol plus cortisone determined simultaneously by HPLC added a new dimension to the diagnosis of Cushing's syndrome. It should be considered when exogenous Cushing's syndrome is suspected or when only one urinary cortisol test is allowed to be ordered.

The objectives of this study were to evaluate (1) the effect of spinal muscle strengthening by loading exercises on the bone mineral density (BMD) of the spine, and (2) the effect of upper extremity loading exercises on the BMD of the midradius and femur in healthy, premenopausal women. The study design was a randomized, controlled trial of 3 years' duration. Ninety-six healthy, premenopausal, white women aged 30-40 years participated; 67 completed the study. All subjects were in good health (normal menses) and were active, but not athletic (that is, not involved in a regular sport activity). Subjects were randomized to an exercise or control group. The exercise group performed a supervised, non-strenuous, weight-lifting exercise program. Exercise performance was supervised once a week at the medical facility. In addition, the subjects performed the exercises twice a week on their own. Dietary calcium intake was to be maintained at 1,500 mg/day in both groups. Bone density was measured at the lumbar spine and hip with dual-energy X-ray absorptiometry at 0, 1, and 3 years. BMD of the midradius was measured with single photon absorptiometry. Measurements of muscle strength were obtained at baseline and every 3 months for 3 years. Maximal oxygen uptake was measured, and the level of physical activity was recorded. Compliance with the exercise program was excellent during the first year of the study, but decreased thereafter. At the end of 3 years, subject withdrawal was about 34% from the exercise group and about 22% from the control group (total subject withdrawal was about 30%). Muscle strength in the exercise group increased significantly at all involved skeletal sites (p values all < 0.001). There was a modest positive correlation between the BMD of Ward's triangle with spinal flexor strength (r = 0.32, p = 0.008) and with grip strength (r = 0.38, p = 0.001). Comparing study groups, we found no significant effect of the loading and nonstrenuous strengthening exercises in the exercise group or free physical activity group (our control group) on BMD at the spine, hip, or midradius measurement sites. In active, but not athletic premenopausal women, additional moderate weight-lifting exercises showed no significant effect on BMD.

Objective: To assess the correlations between plasma concentrations of b-cell polypeptides (insulin, C peptide) and size, multiplicity, and malignancy of insulinomas., Methods: We retrospectively reviewed the medical records of 55 consecutively treated patients at our institution with surgically confirmed insulinoma. The 29 female and 26 male patients ranged from 13 to 77 years of age (median, 46). Plasma insulin, C peptide, proinsulin, and glucose concentrations were analyzed statistically in patients with benign and those with malignant insulinomas., Results: Significant correlations were observed between plasma insulin and tumor size (r = 0.45; P = 0.001) and tumor volume (r = 0.41; P = 0.007) and between plasma C peptide and tumor size (r = 0.38; P = 0.004) and tumor volume (r = 0.33; P = 0.025). No differences between benign and malignant tumors were noted for plasma insulin, C peptide, glucose, nor tumor size or volume. No correlations were found between plasma insulin, C peptide, or glucose and number of tumors., Conclusion: The plasma levels of insulin and C peptide provide qualitative indications about the size of insulinomas.

To assess the mechanism by which estrogen replacement therapy (ERT) enhances renal calcium conservation in perimenopausal women, we studied 18 normal women in early postmenopause before and after 6 months of ERT (cyclic treatment with transdermal estradiol at 100 micrograms/day and medroxyprogesterone acetate at 10 mg/day for the first 12 days of each cycle). The changes after ERT were: serum ionized calcium and ultrafiltrable calcium, no change; serum intact PTH, 38.2% increase (P < 0.0001); serum 1,25-dihydroxyvitamin D, 23.8% increase (P < 0.0001); urinary calcium excretion, 33.3% decrease (P < 0.001); and deoxypyridinoline (a marker for bone resorption), 19.5% decrease (P < 0.0001). Also, ERT increased tubular reabsorption of calcium (TRCa; 97.6% +/- 0.2% to 98.7% +/- 0.1%; P < 0.0001), and this increase correlated with that in serum PTH (r = 0.49; P < 0.05). After the infusion of human PTH-(1-34), the TRCa maximum was greater after ERT than at baseline (99.4% +/- 0.1% vs. 99.0% +/- 0.1%; P < 0.0001), resulting in decreased calcium excretion (0.9 +/- 0.20 vs. 1.43 +/- 0.20 mumol/dL glomerular filtrate; P < 0.001). Thus, in early postmenopause, the major mechanism of increased renal calcium conservation after ERT is an increase in TRCa due to an increase in serum PTH because of estrogen-induced inhibition of bone resorption. However, ERT also may directly increase the TRCa maximum in response to PTH.

PTH has been postulated to play a role in both nocturnal and age-related increases in bone resorption. We tested this hypothesis directly in 10 young (ages 24-35 yr) and 10 elderly (ages 71-78 yr) normal women by measuring the cross-linked N-telopeptide of type I collagen (NTx), a marker for bone collagen breakdown, in 4-h urine collections before and during suppression of PTH secretion by a 24-h iv infusion of calcium. Serum ionized calcium and PTH levels were also measured every 2 h before and during the infusion. In both groups of women, serum PTH levels and urinary NTx excretion followed a circadian pattern before calcium infusion (analysis of variance, P = 0.0001) with peaks in the afternoon and at night for PTH and at night for urinary NTx. During the calcium infusion, the nocturnal urinary NTx excretion peak persisted (P = 0.0001), despite elimination of both PTH peaks. Urinary 24-h NTx excretion (nanomoles per millimoles of creatinine) at baseline was higher in the elderly women (mean +/- SEM, 25.7 +/- 2.1) than in the young women (19.3 +/- 1.7) (P < 0.01), and the decrease during calcium infusion was greater (7.5 +/- 1.9 vs. 4.1 +/- 1.5, P < 0.05). Therefore, the increase in serum PTH levels with age is one of the major factors responsible for the age-related increase in bone resorption. PTH does not mediate the circadian pattern of bone resorption but does play a role in setting the absolute level of bone resorption at which this pattern occurs.

Proinsulin, the precursor of insulin and C-peptide, is detectable in the circulation and is a potential marker of beta-cell dysfunction. Currently, circulating proinsulin can be measured accurately without the interference of insulin or C-peptide by immunometric assays and a few specific radioimmunoassays. An immunochemiluminometric assay was developed in our laboratory by using two immunopurified polyclonal antibodies of C-peptide and insulin from two goats. The C-peptide antiserum was labeled with acridinium ester. The insulin antiserum was immobilized onto a plastic bead. With this assay, proinsulin levels found in noninsulin-treated patients with noninsulin-dependent diabetes mellitus (63 +/- 58 pmol/L; n = 19) were significantly higher (p < 0.05) than levels in insulin-treated patients with noninsulin-dependent diabetes mellitus (30 +/- 24 pmol/L; n = 43), and both were significantly higher (p < 0.001) than proinsulin levels in patients with insulin-dependent diabetes mellitus. In addition, there was a negative correlation between proinsulin levels and duration of years on insulin therapy in patients with noninsulin-dependent diabetes mellitus (r = -0.4795; p < 0.01).

Urinary estrogen levels are important for monitoring the normal pregnancy process as well as for the diagnosis of reproductive diseases. 17 beta-Estradiol and estrone are maintained at very low concentrations in urine and, therefore, are difficult to determine using standard chromatography with UV detection. In the present study, we describe a potential method for the determination of urinary estrogens (estrone, estradiol and estriol) using a solid-phase extraction and rapid capillary electrophoretic (CE) separations. Micellar electrokinetic chromatographic (MEKC) analysis was optimized by evaluating the number of surfactants in a 5 mM borate-5 mM phosphate separation buffer, of which sodium cholate (75-90 mM) was found to be optimal. Changing the hydrophobic character of the separation buffer with organic additives had a significant effect on the resolution of the three estrogens and an internal standard (d-equilenin). The addition of an organic additive (20% acetonitrile) was found to be necessary for the resolution of all components of the mixture. Substitution with 20% methanol provided a similar separation with better resolution but at the cost of increased analysis time. Analysis of two extracted urine samples from 18-weeks and 21-weeks pregnant women showed that, with the present technology, CE can provide adequate resolution and superior speed, but the sensitivity limits attainable with the existing technology may limit its utility to the measurement of estriol and estrone.

Objective: To report the clinical responses and mediator-release profiles of an aspirin-sensitive man with systemic mast cell disease during aspirin desensitization., Material and Methods: We quantified the release of six mediators during aspirin desensitization., Results: Although aspirin was administered cautiously with an initial dose of 20 mg, successful aspirin desensitization necessitated complete monitoring and resuscitation capabilities of a medical intensive-care unit for 4.5 days because of frequent, severe anaphylactoid responses. To our knowledge, this is the first report of a pronounced increase in plasma levels of the vasodilator peptide calcitonin gene-related peptide during episodes of aspirin-induced hypotension. Increases in plasma levels of calcitonin and serum levels of tryptase paralleled those of calcitonin gene-related peptide, but plasma levels of calcitonin remained increased for up to 18 hours. Urinary excretion of histamine and 1-methyl-4-imidazoleacetic acid also showed precipitous, although delayed, increases. Excretion of the prostaglandin D2 metabolite 11 beta-prostaglandin F2 alpha followed a bimodal pattern during aspirin desensitization; after severe hypotensive responses, the maximal value was more than 490,000 pg/mL, but the level decreased to less than 100 pg/mL after therapeutic serum levels of salicylate were attained., Conclusion: These data suggest that the hypotensive responses to aspirin in some patients with systemic mast cell disease may result from the combined effects of several mediators.

Osteoporosis can be predicted to be a new burden to public health in Asia. Currently, the incidence of osteoporosis-related fractures is lower there than in most western communities. By the year 2050, however, 50% of the 6.3 million hip fractures which occur worldwide will be in Asians as a result of an aging population, a decrease in physical activity and westernization of lifestyles. The cost of treatment and cure of these patients will be enormous, a sufficient financial burden to consume current economic gain and cripple the future advancing development of Asian countries. Individual risk factors for osteoporosis have been identified by the extensive Mediterranean Osteoporosis Study (MEDOS). Fortunately, Asians, the rural population and farmers in particular, have the favorable lifestyle identified by the study, including high physical activity and exposure to sunlight. Strikingly, tea drinking, a daily habit in Asia, is also identified as a protective factor against osteoporosis. In addition, bioflavonoids and phytoestrogen-rich soybeans and vegetables are consumed in large quantities by Asians. A soy diet reduces mortality in breast and prostate cancer because it contains weak estrogens. The weakly estrogenic phytoestrogens require further study to demonstrate their pharmacological effect in reducing the rate of osteoporosis. Public health education, however, is needed to encourage the Asian population to maintain their traditionally good lifestyle and to reduce the risk factors for osteoporosis. In turn, these steps may reduce the public health burden by 2050.

Serum PTH concentrations increase with aging and may play an important causal role in age-related bone loss. To better define possible PTH secretory abnormalities with aging, we studied 10 young (aged 27-34 yr) and 10 elderly (aged 71-77 yr) women using sequential infusions of calcium and EDTA. To assess possible age-related resistance of PTH secretion to modulation by 1,25-dihydroxyvitamin D [1,25-(OH)2D], the infusions were repeated after 1 week of oral 1,25-(OH)2D3 therapy (1 microgram/day). Baseline serum intact PTH concentrations were higher in the elderly compared to the young women (mean +/- SEM, 3.8 +/- 0.5 vs. 2.7 +/- 0.4 pmol/L; P = 0.03). In addition, the elderly women had a significantly higher maximal PTH response to hypocalcemia compared to the young women (16.6 +/- 1.1 vs. 12.8 +/- 1.0 pmol/L; P = 0.03). The elderly women also had a greater nonsuppressible component of PTH secretion (0.8 +/- 0.1 vs. 0.4 +/- 0.1 pmol/L; P < 0.001). The set-point for PTH secretion, however, was identical in the elderly and young women (1.18 +/- 0.01 vs. 1.19 +/- 0.01 mmol/L; P = NS). After 1,25-(OH)2D3 administration, both groups had similar reductions in baseline and maximally stimulated PTH levels, indicating that elderly women have normal responsiveness to 1,25-(OH)2D3 suppression of PTH secretion. In addition, maximally stimulated PTH levels in the 1,25-(OH)2D3-treated elderly women decreased to the pretreatment values of young women (13.3 +/- 1.1 vs. 12.8 +/- 1.0 pmol/L; P = NS). thus, elderly women have greater basal, maximal, and nonsuppressible levels of PTH secretion, without alterations in the set-point. These abnormalities are similar to those found in patients with secondary hyperparathyroidism and parathyroid hyperplasia. Further, the abnormal PTH secretory dynamics in elderly women are reversible by short term 1,25-(OH)2D3 therapy.

Objective: To report the recent development and validation of a sensitive 15-minute immunochemiluminometric assay (ICMA) of parathyroid hormone (PTH)., Design: The characteristics and applications of a new "rapid" PTH ICMA are described, and the advantages in comparison with other assays are discussed., Material and Methods: The 15-minute PTH ICMA was achieved by using a higher incubation temperature (45 degrees C rather than room temperature) and a concentration of tracer antibodies that was 5 times that in the regular 18-hour assay. The sensitivity, recovery, and linearity of dilution of the rapid ICMA were assessed, as was its ability to reflect intraoperative changes in PTH concentration after parathyroidectomy in patients with hyperparathyroidism., Results: The sensitivity of the ICMA was 0.3 pmol/L in comparison with 0.08 pmol/L for the regular 18-hour assay (normal range, 1.0 to 5.0). The recovery of added PTH was 84% by using ethylene-diaminetetraacetic acid-plasma as the specimen. Moreover, the linearity of specimens after dilution was good. The assay demonstrated a rapid response to parathyroid gland removal. In most patients, PTH levels declined precipitously (to less than 30% of basal values) within 5 minutes after parathyroid resection. PTH levels remained low to undetectable at 10 and 20 minutes after excision., Conclusion: This rapid PTH ICMA may be of assistance to endocrine surgeons in the monitoring of PTH function after both parathyroid and thyroid surgical procedures.

We used a newly developed immunochemiluminometric assay of proinsulin to determine its relative utility vis-à-vis C-peptide and insulin for the diagnosis of insulinoma. The evaluation was conducted in 20 consecutive patients with histologically confirmed insulinoma and 22 normal subjects who underwent a prolonged fast according to a standard protocol. Patients with insulinoma fasted to the point of demonstrating Whipple's triad; normal subjects fasted to 72 h. At the end of the prolonged fast, when the glucose value was 2.8 mmol/L or less (50 mg/dL), all three hormones had equal sensitivity (100%) in detecting insulinoma with no overlap with the values of normal subjects. When glucose levels were between 2.8 mmol/L (50 mg/dL) and 3.3 mmol/L (60 mg/dL) at the end of the prolonged fast, proinsulin was better than C-peptide and insulin in the diagnosis of insulinoma. The sensitivity was 90% for proinsulin and 85% for both C-peptide and insulin. Therefore, proinsulin not only is useful for the diagnosis of insulinoma, but it may have greater diagnostic accuracy than C-peptide and insulin.

Background: Although recent investigations report the elevation of plasma endothelin (ET) in congestive heart failure (CHF), it remains unclear if this elevation is that of the biologically active peptide ET-1 or of its precursor big-ET. Furthermore, it is unclear if such elevation is associated with increased myocardial ET and if the molecular form from cardiac tissue is altered ET. Last, it remains to be established whether circulating ET is increased at the earliest stage of CHF in patients with asymptomatic left ventricular dysfunction and correlates with the magnitude of ventricular dysfunction., Methods and Results: The present study was designed to investigate concentrations and molecular forms of ET in plasma and cardiac tissue in healthy subjects and CHF patients with New York Heart Association (NYHA) class I through IV using cardiac radionuclide angiogram, cardiac myocardial biopsy, radioimmunoassay, gel permeation chromatography (GPC), and immunohistochemical staining (IHCS). Plasma ET was increased only in patients with moderate (NYHA class III) or severe (NYHA class IV) CHF compared with healthy subjects and individuals with asymptomatic (NYHA class I) or mild (NYHA class II) CHF. The elevation of circulating ET in CHF showed a negative correlation with left ventricular ejection fraction and cardiac index and a positive correlation with functional class and left ventricular end-diastolic volume index. GPC demonstrated that immunoreactive plasma ET was ET-1 in healthy subjects and both mature ET-1 and its precursor big-ET in severe CHF patients, with big-ET the predominant molecular form. Cardiac tissue concentrations and IHCS revealed ET presence in healthy atrial and ventricular tissue, which were not different in severe CHF. GPC revealed that the molecular form of cardiac ET was ET-1 in both healthy and CHF hearts., Conclusions: The present study establishes for the first time that the elevation of plasma ET in severe human CHF represents principally elevation of big-ET. Second, ET is present in healthy and failing myocardia, and its activity by both immunohistochemistry and radioimmunoassay is not changed in CHF. Furthermore, the elevated plasma ET is characteristic of severe CHF and not asymptomatic or mild CHF. In addition, the degree of plasma elevation of ET correlates with the magnitude of alterations in cardiac hemodynamics and functional class. The present study confirms and extends previous investigations of ET in human CHF and establishes the evolution of circulating and local cardiac ET in the spectrum of human CHF.

Insulin-like growth factor-I (IGF-I) levels in plasma were measured in healthy twin children. The within-pair correlation for 43 monozygotic pairs was r = 0.91 (P < or = 0.0001), an association significantly higher than that for same sex dizygotic pairs (r = 0.40; P < or = 0.06). The high correlation for monozygotic twins indicated a marked genetic influence on IGF-I levels. After correction for age and sex, the correlation between IGF-I level and height was r = 0.38 (P < or = 0.0001). These findings provide clear evidence that IGF-I levels correlate with height, a growth characteristic known to be genetically controlled.

Insulin-like growth factors are potent mitogens for breast cancer cell proliferation. This effect is modulated by the circulatory and extracellular IGFBPs as well as by the affinity of ligand binding receptors on the target cells. Antiestrogens have been shown to reduce both circulatory and microenvironmental IGF levels and thus suppress the IGF-I-induced growth of both ER-positive and ER-negative breast cancer cells. However, the effects of antiestrogens in down regulation of type I IGF receptor and in altering the autophosphorylation tyrosine kinase activity of EGF receptors are mainly observed in ER-positive cells. Furthermore, alteration of IGFBP by antiestrogens such as a marked increase of IGFBP-I production have been shown to inhibit the proliferative effect of IGF-I on ER-positive, but stimulate this effect, on ER-negative cells. Such differential effects from IGF receptor and IGFBP may explain the clinical outcome that tumor regression from antiestrogens is mainly observed in ER-positive type. This assumption based on IGF regulation alone is certainly an oversimplistic view amid the complexity of autocrine, paracrine, and endocrine functions.

An extraction radioimmunoassay of calcitonin (CT) was used to replace the direct radioimmunoassay, in which false-negative results (normal basal CT values but above normal ["positive"] results after pentagastrin stimulation) occurred in 26 to 30% of cases. During the past 6 years, 170 pentagastrin stimulation tests were performed in 135 patients at our institution. We reviewed the results to determine whether extraction radioimmunoassay yielded improvement. Of the 135 patients, 6 (4.4%) had indeterminate results of pentagastrin stimulation tests that consisted of normal basal levels but increased values after pentagastrin stimulation, a considerable improvement over the results with direct assay. Six other patients had indeterminate results of pentagastrin testing--increased basal CT levels but normal values after pentagastrin stimulation. Indeterminate results of pentagastrin tests occurred primarily in postoperative or preoperative patients with a family history of medullary thyroid carcinoma (MTC). The reasons for the indeterminate results were unclear, as was their importance as a prelude to disease, but two preoperative patients with indeterminate results eventually had positive results and were found to have MTC at operation. We conclude that extraction radioimmunoassay is more reliable than direct radioimmunoassay for the diagnosis of MTC. Determination of a basal CT value and of the CT value after pentagastrin stimulation is sufficient for diagnosing MTC.

Background: beta-Atrial natriuretic factor (beta-ANF) is an antiparallel dimer of alpha-ANF (alpha-ANF) with diminished cyclic GMP generation in vitro. To date, the presence of beta-ANF in the circulation of humans with severe congestive heart failure (CHF) remains controversial. The current study was designed to determine the presence and magnitude of circulating beta-ANF in severe CHF, to correlate plasma beta-ANF with the degree of ventricular dysfunction, and to investigate the role of human plasma and atrial tissue in the degradation of beta-ANF., Methods and Results: Venous plasma samples were obtained from patients (n = 12) with severe CHF and normal volunteers (n = 8). Total plasma ANF was measured by radioimmunoassay. alpha-ANF and beta-ANF in nonextracted plasma were separated by gel filtration chromatography using a P-6 column. Right atrial tissue samples (n = 5) were collected from a different group of patients at the time of open-heart surgery. 125I beta-ANF and I125 ANF were incubated with atrial tissue or plasma. The corresponding peak areas of beta-ANF were determined by Tamaya Digital Planimeter. beta-ANF represented 61% of total plasma ANF in CHF patients and was not detected in normal human plasma. The elevation of beta-ANF correlated with the severity of ventricular dysfunction. Thirty percent of beta-ANF and 100% alpha-ANF were converted to smaller peptide fragments in atrial tissue no conversion in plasma., Conclusions: beta-ANF is the principal form of circulating ANF in patients with severe CHF and correlates with the degree of left ventricular dysfunction. beta-ANF is not generated from alpha-ANF and may be degraded rapidly in atrial tissue to smaller peptide fragments that do not occur in plasma. As beta-ANF is reported to have reduced biological action, the current studies may support the conclusion that the ANF system in CHF has reduced functional activity despite increases in circulation concentrations.

The efficacy of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and the new analogue calcipotriene (MC 903) in the treatment of psoriasis was investigated. Eight patients with psoriasis were enrolled in a pilot study with systemically administered vitamin D3 and were given 1,25(OH)2D3 (Rocaltrol; Hoffmann-La Roche) in dosages from 0.5 microgram to 2 micrograms/day for a 6-month trial. In one patient, the psoriatic plaques resolved within 2 months after treatment (0.5 microgram/day) was initiated. Moderate improvement was noted in one other patient (1 microgram/day). The serum level of 1,25(OH)2D before treatment was not less than the normal range of the adult population. Side effects of systemically administered 1,25(OH)2D3 included a dose-dependent increase in the 24-hour urinary calcium excretion and a decrease in the total number of platelets. Seven patients with symmetric plaque-type psoriasis were treated topically with 2 micrograms/g of 1,25(OH)2D3 in petrolatum. During 3 months of follow-up, mild improvement was noted in three patients. Five patients in the calcipotriene study were part of a nationwide double-blind placebo-controlled trial by Bristol-Myers Squibb. Moderate to marked improvement was noted in the two patients who received 50 micrograms/g of calcipotriene topically. The three patients who received placebo showed no response. We conclude that a subset of patients with psoriasis responds well to 1,25(OH)2D3. Calcipotriene is efficacious and an excellent alternative to topically applied corticosteroids.

We developed a competitive chemiluminoimmunoassay of osteocalcin that is similar to radioimmunoassay but uses acridinium-ester-labeled antigen instead of 125I-labeled osteocalcin; a second antibody immobilized on plastic beads is used to separate free and bound fractions. There was good correlation of the new chemiluminoimmunoassay (y) with a polyclonal antiserum (R102) radioimmunoassay (x) used in many previous clinical studies (r = 0.96, y = 0.968x + 2.69, Sy/x = 0.029, n = 86). The new assay recognized both the intact and the small fragment of osteocalcin in plasma and detected decreases of them (total, approximately 47%) after a 24-h infusion of parathyroid hormone. Patients with primary hyperparathyroidism had increased concentrations of intact osteocalcin. Children had higher concentrations of osteocalcin than adults did. Healthy women had greater osteocalcin concentrations at ages 50-70 years than earlier. Inverse correlations of bone mineral density and osteocalcin were found in healthy women and in women with osteoporosis.

Antibodies to ACTH1-24 detected by radioimmunoassay were present in the serum of a child with opsoclonus-myoclonus for at least 24 weeks after discontinuation of chronic ACTH treatment. The antibody-bound ACTH did not interfere with cortisol secretion. Six other children with opsoclonus-myoclonus and 16 control sera, including patients with chronically elevated endogenous ACTH, did not exhibit autoantibodies to ACTH. Antibodies to ACTH should be sought in patients who develop tolerance to ACTH treatment. The indirect but not direct ACTH assay method is sensitive to the presence of ACTH antibodies.

C-Peptide, a marker for insulin secretion, is purported to be elevated in patients with insulinoma but diagnostic criteria have not been established. Thirty-seven patients with histologically confirmed insulinoma studied preoperatively, 19 normal subjects, and 2 patients who subsequently acknowledged self-administration of insulin underwent the prolonged fast (< or = 72 h) according to a standard protocol. Plasma glucose, C-peptide, and insulin were measured every 6 h until plasma glucose was less than or equal to 3.3 mmol, then hourly until Whipple's triad was demonstrated or until 72 h without symptoms was reached. At the termination of the fasts, plasma was analyzed for sulfonylurea. Statistical analysis was by rank sum test. Data are expressed as median (range). The durations of fasts were 20 (2.5-68) h for patients with insulinomas and 72 h for normal subjects. At the end of fasts plasma glucose, C-peptide, and insulin concentrations were 2.2 (1.4-2.9) vs. 3.6 (2.7-5.5) mmol, P < 0.001; 0.60 (0.20-1.92) vs. 0.13 (0.07-0.43) nmol, P < 0.001; and 126 (35-840) vs. 35 (35-126) pmol, P < 0.001, respectively, for insulinoma patients and normal subjects. All plasma samples were negative for sulfonylurea. Insulinoma patients had C-peptide values at the end of the fasts greater than or equal to 0.20 nmol whereas normal subjects and patients with insulin factitial hypoglycemia had C-peptide concentrations less than or equal to 0.10 nmol when plasma glucose was less than or equal to 2.8 mmol. Insulinoma is confirmed in a sulfonylurea negative patient with Whipple's triad during the prolonged fast and a concomitant C-peptide concentration greater than or equal to 0.20 nmol.

PTH clearly plays a role in maintaining the hypercalcemia of familial benign hypercalcemia (FBH or familial hypocalciuric hypocalcemia). To better define the abnormalities of parathyroid function in FBH and primary hyperparathyroidism (1 degree HPT), we used a two-site immunochemiluminometric assay for intact PTH to examine PTH suppressibility in normal individuals and patients having FBH or 1 degree HPT. Twelve normal, 11 FBH, and 7 1 degree HPT subjects were given calcium (Ca) iv with frequent sampling for ionized Ca and intact PTH. In normal and FBH subjects, plasma PTH levels decreased essentially identically in response to iv Ca. In the 1 degree HPT group, PTH was not normally suppressible. However, there was a spectrum of responsiveness in 1 degree HPT patients, with a significant correlation between tumor mass and degree of PTH nonsuppressibility (r = 0.87, P = 0.01). Analysis of the relationship between plasma PTH and ionized Ca values in the three groups demonstrated a shift to the right in the FBH curve, with no difference of slope, consistent with the notion of a simple "set-point" error in FBH. In contrast, the curve in 1 degree HPT was not only shifted to the right but also differed from normal in slope (normal, -8.92; 1 degree HPT, -3.92, P = 0.04). Thus, we propose that the parathyroid functional abnormality in FBH represents a simple set-point error, whereas the defect in 1 degree HPT consists of a set-point error combined with varying degrees of Ca nonsuppressible PTH secretion that may be related to tumor mass.

In this study, we reviewed the diagnostic efficiency of laboratory tests that are performed for assessment of patients with Cushing's syndrome or adrenal insufficiency. Baseline laboratory data from patients subsequently diagnosed with adrenal dysfunction were analyzed for tests performed between 1987 and 1989 at our institution. Results were analyzed for 36 patients diagnosed with pituitary-dependent Cushing's syndrome, 15 with ectopic Cushing's syndrome, 12 with adrenal-dependent Cushing's syndrome, 20 with primary adrenal insufficiency, and 7 with secondary adrenal insufficiency. Tests reviewed were plasma cortisol, plasma corticotropin, urinary free cortisol, urinary 17-ketosteroids, urinary ketogenic steroids, low-dose and high-dose dexamethasone suppression, and metyrapone stimulation. Our findings suggest that a substantial proportion of diagnoses could be based on the results of three tests--plasma corticotropin, plasma cortisol, and urinary free cortisol. We present a nomogram that combines the results of plasma corticotropin and plasma cortisol testing to enhance the diagnostic efficiency of these tests.

Two goats were immunized with synthetic C-peptide. Their antisera were immunopurified separately on a C-peptide affinity column. The purified antibodies from one goat were labeled with acridinium ester; the other goat's antibodies were immobilized on plastic beads. Standards were synthetic C-peptide. The new immunochemiluminometric assay (ICMA) for C-peptide was compared with our routine radioimmunoassay (RIA) by using Novo's 125I-labeled C-peptide and Cambridge Medical's antiserum. Good correlations were found between the new ICMA and the RIA (r = 0.951; ICMA = 1.07 RIA + 147; n = 112). The new ICMA showed good recoveries (91 percent to 108 percent) of added C-peptide and parallelism of diluted specimens. The incubation time was shortened from 48 hours for RIA to five hours for ICMA. In addition, it was shown that polyclonal antisera from two animals immunized by the same antigen and purified by the same affinity column can be used to prepare immunometric assays.

The performance and clinical utility of an automated assay of intact parathyroid hormone (parathyrin, PTH) are evaluated. The method is based on the extraction of PTH from plasma by an HPLC column containing immobilized anti-(44-68 PTH) antibodies. The PTH retained is detected with a postcolumn reactor and use of anti-(1-34 PTH) chemiluminescent-labeled antibodies. The total cycle time of the assay is 6.5 min per injection after a 1-h incubation. The lower limit of detection for PTH in a 66-microL plasma sample was 0.5 pmol/L based on peak heights and 0.2 pmol/L based on peak areas. Mean analytical recovery for PTH added to plasma was 97%. The within-day precisions (CVs) for 4.2 and 30 pmol/L PTH plasma samples were 9.2% and 5.6% and the day-to-day precisions were 10.3% and 5.7%, respectively. No significant interferences from 1-34, 44-68, or 53-84 PTH fragments were noted, even at highly increased concentrations of fragments. The correlation of results with those of a manual assay of intact PTH was 0.97, and the results showed good agreement with disease state for patients with hypo- or hyperparathyroidism. The specificity of the assay for primary hyperparathyroidism was greater than 95%. We discuss the advantages (speed and quality control) of this approach over current immunoassays and the potential use of this method for detecting other analytes.

Three immunometric assays of parathyroid hormone (PTH)--a commercial immunoradiometric assay, an in-house immunoradiometric assay, and an immunochemiluminometric assay--were evaluated in 50 patients with surgically proven primary hyperparathyroidism. Of these patients, 43 had increased values with the commercial assay (sensitivity, 86%), whereas 45 patients had increased concentrations with both the in-house immunoradiometric and the in-house immunochemiluminometric assays (sensitivities, 90%). Because of the results of this comparison study, we confidently chose the immunochemiluminometric assay as our routine assay; this assay was evaluated retrospectively in 361 patients with surgically proven primary hyperparathyroidism. In 45 patients, PTH values were below the upper limit of normal (sensitivity, 88%). The results indicate that the sensitivities of current immunometric assays are approximately 90%. Twenty patients who had hypercalcemia associated with malignant involvement were assessed with the immunochemiluminometric assay. Of these 20 patients, 19 had subnormal PTH values, and 1 had a value within the normal range. In contrast, in the past, PTH values determined with radioimmunoassays have often been in the normal range for such patients. Thus, an immunometric PTH assay is superior to a radioimmunoassay in the differential diagnosis of hypercalcemia associated with malignant disease.

The immunochemiluminometric assay described uses immobilized anti-human parathyrin (parathyroid hormone, hPTH)(1-44) and anti-hPTH(44-68) antisera and acridinium ester-labeled anti-hPTH(1-34) to simultaneously measure both intact hPTH and its amino-terminal fragments. Results by the assay correlate well with those by a cAMP-based bioassay and the Nichols Allegro immunoradiometric assay. The minimal detection limit is 0.08 pmol/L. The normal range is 1.0-5.0 pmol/L, and values are higher in older women. About 90% of study patients with surgically proven parathyroid adenomas had above-normal preoperative PTH concentrations, whereas patients with hypercalcemia of malignancy had normal or suppressed values. This assay was designed to detect both intact PTH and amino-terminal PTH fragments; however, chromatographic fractionation of pools of primary and secondary hyperparathyroid plasma showed virtually no amino-terminal fragment activity. Nonetheless, the design is important because the absence of carboxyl-terminal binding sites prevents interference by carboxyl-terminal fragments and because bioactive amino-terminal fragments will react in the assay if they are present in the patients' sera or if they are produced by in vitro proteolysis of intact PTH.

An in-house method was used, including dissociation of Somatomedin C/insulin-like Growth Factor I (Sm-C/IGF-I) binding proteins with acid and extraction of Sm-C/IGF-I by C-2 cartridge before radioimmunoassay, to measure plasma Sm-C/IGF-I levels in three groups of patients--total of 19--with acromegaly who underwent transsphenoidal removal of pituitary tumors. In group A (n = 7), the Sm-C/IGF-I levels did not decrease to normal on postoperative day 1 even though the growth hormone concentration had decreased to normal. In group B (n = 4), the Sm-C/IGF-I level decreased to normal in three of the four patients on postoperative day 4, but it remained elevated in one. At two months postoperatively, all four patients had normal values. Group C patients (n = 8) had had an operation two to 23 years previously, and all remained in remission with normal Sm-C/IGF-I levels. Our findings indicate that the Sm-C/IGF-I assay is useful for monitoring acromegaly after transsphenoidal removal of pituitary tumors, but it should be ordered at least four days postoperatively.

To assess the effects of gender, age, and body mass index (BMI) on suppression of plasma C-peptide during insulin-induced hypoglycemia, 101 lean and obese, healthy men and women ages 20 to 80 yr underwent infusion of human regular insulin, 0.125 U/kg over 60 min after an overnight fast. Plasma glucose, insulin, and C-peptide were measured every 30 min for 120 min. C-peptide concentrations were influenced by gender at 30 min, by BMI at baseline and both BMI and age at all subsequent time points. Because of variations in baseline plasma C-peptide concentrations, percent decrease in C-peptide was evaluated. Significantly less percent decrease of C-peptide with increased age at 30, 60, and 90 min and with increased BMI at 30 and 60 min were noted with no effect of gender. From stepwise regression analysis using multiple, additional variables only the plasma glucose concentration at 30 min made a significant, albeit small (8%), contribution to the variability in percent decrease in C-peptide at 60 min. When C-peptide responses from eight histologically confirmed insulinoma patients were contrasted to values adjusted for age, gender, and BMI of normal subjects, all insulinoma patients had abnormal responses when percent decrease in C-peptide was used, whereas only four insulinoma patients had abnormal response when actual C-peptide concentrations were used.

An automated sandwich immunoassay was developed based on high-performance immunoaffinity chromatography and chemiluminescent detection, using the determination of parathyroid hormone (PTH) in plasma as a model system. In this method, injections of plasma and acridinium ester-labeled anti-(1-34 PTH) antibodies were made onto a column containing immobilized anti-(44-68 PTH) antibodies. Upon elution, PTH and its associated labeled antibody were combined with an alkaline peroxide postcolumn reagent, and the resulting light production was measured. Factors considered in optimizing this system included the column's dissociation properties, the rate of light production in the postcolumn reactor, and the use of sequential vs simultaneous injection of sample and labeled antibody. The final system developed required 6 min per plasma injection, following a 1-h incubation of sample with labeled antibody. The response was linear over 2-3 orders of magnitude and the lower limit of detection for a 66-microL plasma sample was only 16 amol, or 2.4 x 10(-13) M. Overall, this method had a precision and response similar to those of manual PTH methods but required 24-fold less time to perform. By using different immobilized and labeled antibodies, this method could easily be adapted for use with other analytes.

The cause of hypercalcemia in familial benign hypercalcemia (FBH; also called familial hypocalciuric hypercalcemia) is unclear, although it is PTH dependent. It is also uncertain how plasma PTH levels are related to the severity of biochemical abnormalities in FBH. Because the PTH-related peptide (PTHrP) has many PTH-like actions, it might have a role in the hypercalcemia of FBH. Thus, we studied 29 patients with FBH from 11 families, 29 age- and sex-matched controls, and 42 patients with primary hyperparathyroidism (1 degree HPT), measuring PTH with a highly sensitive two-site immunochemiluminometric assay and the hypercalcemic tumor factor PTH-related peptide (PTHrP) with an extraction/concentration RIA. Plasma PTH values were elevated in 86% of 1 degree HPT patients (36 of 42), but in only 20% of FBH patients, (6 of 29). Plasma PTHrP was elevated in 1 FBH patient, and the group mean value was normal. Plasma PTH was positively correlated with calcium (Ca) in 1 degree HPT (r = 0.66; P less than 0.0001) and in FBH (r = 0.53; P less than 0.004), but the slopes of the regressions were markedly different: 1 degree HPT, 6.72; FBH, 1.61 (P less than 0.0001). There was a negative correlation between PTH and phosphorus (P) in 1 degree HPT (r = -0.39; P less than 0.01) and in FBH (r = -0.41; P less than 0.03), but, again, the slopes differed greatly: 1 degree HPT, -6.57; FBH, -1.95 (P less than 0.0001). There were no correlations between PTHrP and Ca or between PTH and PTHrP. The sums and products of PTH and PTHrP were not better correlated with Ca than PTH alone. Thus, PTH values are lower at given Ca and P levels in patients with FBH than in those with 1 degree HPT, suggesting that PTH is more effective in raising Ca and lowering P in FBH than in 1 degree HPT. The enigma of FBH remains: what molecular defect can simultaneously cause parathyroid cell insensitivity to Ca, enhanced renal tubular reabsorption of Ca, increased renal rejection of P, and enhanced or retained sensitivity to PTH?

There is doubt about concentrations of circulating calcitonin gene-related peptide (CGRP) and the value of plasma CGRP measurements in the detection and follow-up of medullary thyroid carcinoma (MTC). Thus, we developed an immunochemiluminometric sandwich assay for CGRP using antibodies purified from a polyclonal antiserum against human CGRP. The assay was sensitive (limit of detection, 0.4 pmol/L; multiply by 3.7892 to derive nanograms per L) and highly specific [no cross-reaction with human calcitonin (CT)]. Normal plasma CGRP values ranged from less than 0.4 to 4.5 pmol/L (median, 0.8; n = 31), with 61% having detectable levels. Values in samples from patients with MTC were elevated: unoperated patients (n = 10), 4.7-137 pmol/L (median, 7.1); and operated patients with gross persistent or recurrent tumor (n = 14), 4.7-171 pmol/L (median, 23.2). In contrast, CGRP values were normal in 78% of nine postoperative patients with elevated CT, but no detectable tumor (range, less than 0.4 to 6.3 pmol/L; median, 1.6). CGRP levels increased after pentagastrin injection in MTC patients, but less than did CT values. Cultured MTC cells in vitro secreted large amounts of CGRP, and rat nerve root ganglia, human osteoblasts, and microvessel endothelial cells secreted lesser amounts. We conclude that CGRP circulates in normal plasma, but at very low levels. Plasma CGRP concentrations are frequently high in patients with MTC, but primarily in those with gross tumor or metastases. Plasma CT assay is the preferable test for MTC, but CGRP assay deserves prospective study for a possible role in predicting gross metastasis.