One-step direct assay for mature-type adrenomedullin with monoclonal antibodies

Adrenomedullin (AM) is a potent hypotensive peptide. Plasma contains mature-type AM (m-AM), which is amidated at the carboxy terminus, and an intermediate, AM-Gly. We developed a one-step two-site IRMA specific for determining human m-AM with monoclonal antibodies. The detection limit was 0.5 pmol/L, and the working range (CV <15%) was 1-300 pmol/L. Dilution of plasma samples showed good linearity. The recovery of added AM was 91-118%. The intra- and interassay imprecision values (CVs) were 4.4-8.2% and 5.5-8.3%, respectively. The assay had no cross-reactivity with AM-Gly or other peptides similar to AM. The mean (+/- SD) plasma human m-AM concentration of 61 healthy subjects was 1.18 +/- 0.65 pmol/L. In conclusion, our IRMA makes it possible to specifically measure m-AM, using a small amount of plasma sample (0.2 mL) by a one-step overnight assay without prior extraction. Our simplified method would be suitable for clinical studies on AM, especially when large numbers of samples must be processed.     

Circulating N-terminal atrial natriuretic peptide as a marker for symptomless left-ventricular dysfunction

Early identification of patients with symptomless left-ventricular dysfunction and early pharmacologic intervention may have an impact on the outlook of patients with heart failure. Atrial natriuretic peptide (ANP) is a cardiac hormone that is released as a C-terminal (C-ANP) and an N-terminal peptide (N-ANP). Since N-ANP has reduced clearance rates compared with C-ANP, N-ANP circulates at higher concentrations. Based on the known increased concentration of C-ANP in symptomatic congestive heart failure, our study was designed to evaluate prospectively N-ANP profile and left-ventricular function in subjects with symptomless and symptomatic heart failure, and the role of plasma N-ANP as a marker for early identification of patients with heart failure. 180 patients who were referred for rest and exercise radionuclide angiography for evaluation of left-ventricular function were studied. Blood was taken for measurement of C-ANP and N-ANP before angiography. Patients were grouped according to New York Heart Association (NYHA) heart failure classification and left-ventricular function. Mean (SD) plasma N-ANP concentration in patients with symptomless left-ventricular dysfunction (NYHA class I, n = 70) was 243 (256) pmol/L (range 27-922 pmol/L), and was higher (p < 0.001) than in 25 control subjects (28 pmol/L). A plasma N-ANP concentration above 54 pmol/L (mean +/- 1.96SD of the control group) had a sensitivity of 90% and a specificity of 92% for detection of patients with symptomless left-ventricular dysfunction. We have shown that plasma N-ANP concentrations are significantly increased in patients with symptomless left-ventricular dysfunction and that this peptide can serve as a marker for diagnosis of such patients.     

The porphyromonas gingivalis prtP/kgp homologue exists as two open reading frames in strain 381

Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels increase in patients with heart failure with the progression of clinical symptoms and with the deterioration of hemodynamics; consequently, assay methods for these peptides may be useful in the follow-up of cardiac patients. Non-competitive immunoradiometric assay (IRMA) methods for ANP or BNP do not generally require preliminary extraction and/or purification of the plasma sample, and so may be more suitable than competitive immunoradiometric assay (RIA) methods for the routine assay of plasma peptide concentrations. We evaluated the analytical characteristics and clinical usefulness of two IRMAs for plasma ANP and BNP, to verify whether these methods may be considered suitable for the follow-up of patients with heart failure. Both methods are based on the solid-phase sandwich IRMA system, which uses two monoclonal antibodies prepared against two sterically remote epitopes of peptide molecule; the first antibody was coated on the beads solid-phase and the second was radiolabeled with 125I. Blood samples were collected from a brachial vein in ice-chilled disposable polypropylene tubes containing aprotinin and EDTA after the patient had rested for at least 20 min in the recumbent position. Plasma samples were immediately separated by centrifugation and stored at -20 C until assay. The IRMA methods showed a better sensitivity and a wider working range sensitivity (about 2 ng/l) than those of RIA methods. Moreover, the normal range found with these methods (ANP = 16.1 +/- 8.6 ng/l, 5.2 +/- 2.8 pmol/l, BNP = 8.6 +/- 8.2 ng/l, 2.5 +/- 2.4 pmol/l) was similar to that generally reported using the most accurate methods, such as the other IRMAs or RIAs, using a preliminary extraction and purification of plasma samples with chromatographic procedures. Our results obtained in patients with different degrees of heart failure indicate that plasma ANP and BNP increase with the progression of clinical symptoms (NYHA class) (ANOVA p < 0.0001). Indeed, circulating levels of ANP (R = -0.701, no. = 86) and BNP (R = -0.745, no. = 55) were significantly (p < 0.0001) and negatively correlated with the left ventricular ejection fraction values. Furthermore, a close curvilinear regression (R = 0.960, no. = 215) was found between ANP and BNP values, because plasma BNP progressively increases more than plasma ANP in patients with different stages of heart failure. In conclusion, IRMA methods are preferable for the measurement of plasma ANP and BNP for experimental studies and routine assay because they are more practicable, sensitive and accurate than RIA procedures. Finally, BNP assay appears to be better than ANP for discriminating between normal subjects and patients with different degrees of heart failure.     

Rapid assay of plasma brain natriuretic peptide in the assessment of acute dyspnoea

AIM: Recognition of heart failure may be difficult in patients presenting with acute dyspnoea, particularly in the presence of chronic airways obstruction or obesity. In a previous study of patients with acute dyspnoea, we showed that the measurement of plasma brain natriuretic peptide (BNP)-a hormone secreted in increased amounts by the failing heart-accurately distinguishes heart failure from primary lung disorder. The aim of the present study was to develop a rapid assay for BNP and evaluate its diagnostic use in patients acutely hospitalised for increasing dyspnoea of any cause. METHODS: A rapid assay for plasma BNP, providing results within 24 h of blood collection, was developed without loss of precision. The results of the rapid and previously established BNP assays were highly correlated (r = 0.9). To determine the diagnostic value of the rapid assay, measurements were undertaken on the day of admission in 123 breathless patients (mean age 68.3, range 23 to 90 years) and related to conventional diagnostic assessments and final outcome. RESULTS: In patients diagnosed and treated urgently for clinical heart failure, plasma BNP was significantly higher (115 (SE 13) pmol/L, n = 39) than in those without clinical heart failure (33 (5) pmol/L, n = 84, p < 0.001). Using a cut-off of 50 pmol/L for the presence of heart failure, there was discordance between BNP level and clinical diagnosis in 21 of 123 cases. Reassessment after independent analysis of discordant cases increased the difference in BNP level in the presence (123 (13) pmol/L, n = 43) or absence (24 (1.5) pmol/L, n = 80) of heart failure. Using two way analysis of variance, no further improvement in discrimination was found when chest radiographs were used together with the BNP data. CONCLUSION: Rapid BNP assays are practicable and provide accurate information on cardiac status-superior to chest radiographs in many cases-early in the course of the patient's presentation with acute dyspnoea.     

Hyperproduction of erythropoietin in nonanemic lead-exposed children

The plasma concentration of N-terminal atrial natriuretic peptide (N-ANP) has been shown to be predictive of both clinical status and survival in patients with heart failure. In this analysis the relationship between N-ANP, morbidity and hospitalization time was evaluated in 417 patients with stable, congestive heart failure recruited from an active, outpatient heart failure registry. Hospital admissions along with the duration of stay occurring after the initial N-ANP sampling during the period of data collection were recorded. A total of 755 admissions occurred, accounting for 7917 days' hospitalization. Relative hospitalization times (in-hospital days/observation period) per N-ANP quartiles I-IV were: 1.2 (+/- 2.7)%, 5.5 (+/- 12.2)%, 10.0 (+/- 21.5)% and 20.8 (+/- 34.3)%, respectively. Although N-ANP levels were correlated with age (r = 0.234, p < 0.0001), division by age quartiles did not significantly predict relative hospitalization times. These data indicate that the degree of cardiac endocrine activation and subsequent N-ANP release is related to morbidity in patients with heart failure and that moderate elevation in N-ANP levels is associated with a substantially increased hospitalization time. N-ANP sampling should be of value as a supplement to clinical evaluation in the assessment of the individual patient with this common syndrome.     

Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin: new neurohormonal predictors of left ventricular function and prognosis after myocardial infarction

BACKGROUND: Newly discovered circulating peptides, N-terminal pro-brain natriuretic peptide (N-BNP) and adrenomedullin (ADM), were examined for prediction of cardiac function and prognosis and compared with previously reported markers in 121 patients with myocardial infarction. METHODS AND RESULTS: The association between radionuclide left ventricular ejection fraction (LVEF) and N-BNP at 2 to 4 days (r=-.63, P<.0001) and 3 to 5 months (r=-.58, P<.0001) after infarction was comparable to that for C-terminal BNP and far stronger than for ADM (r=-.26, P<.01), N-terminal atrial natriuretic peptide (N-ANP), C-terminal ANP, cGMP, or plasma catecholamine concentrations. For prediction of death over 24 months of follow-up, an early postinfarction N-BNP level > or = 160 pmol/L had sensitivity, specificity, positive predictive value, and negative predictive values of 91%, 72%, 39%, and 97%, respectively, and was superior to any other neurohormone measured and to LVEF. Only 1 of 21 deaths occurred in a patient with an N-BNP level below the group median (Kaplan-Meier survival analysis, P<.00001). For prediction of heart failure (left ventricular failure), plasma N-BNP > or = 145 pmol/L had sensitivity (85%) and negative predictive value (91%) comparable to the other cardiac peptides and was superior to ADM, plasma catecholamines, and LVEF. By multivariate analysis, N-BNP but not ADM provided predictive information for death and left ventricular failure independent of patient age, sex, LVEF, levels of other hormones, and previous history of heart failure, myocardial infarction, hypertension, or diabetes. CONCLUSIONS: Plasma N-BNP measured 2 to 4 days after myocardial infarction independently predicted left ventricular function and 2-year survival. Stratification of patients into low- and high-risk groups can be facilitated by plasma N-BNP or BNP measurements, and one of these could reasonably be included in the routine clinical workup of patients after myocardial infarction.     

Luminometric single step urea assay using ATP-hydrolyzing urease

An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 micromol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (v(blank)) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96-103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2-21.8 mmol/L) were 3.6-8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02-1.03 and intercepts of 0.08-0.23 mmol/L.     

Radioimmunoassay of cholecystokinin in human plasma

A sensitive radioimmunoassay for cholecystokinin (CCK) has been developed. Porcine CCK-33 was labelled by conjugation with 125I-hydroxyphenyl-propionic acid succinimide ester. Antibodies were raised against porcine CCK-33 covalently coupled to egg albumin. Plasma samples were extracted with 96% ethanol prior to assay. Free and bound hormone were separated by dextran-coated charcoal. The antibodies bound CCK-8 and CCK-33 with equimolar potency. The assay detection limit was 1 pmol/l plasma. Within and between assay coefficients of variation were +/- 12.7 and 13.0% at mean plasma CCK concentrations of 13.2 and 13.6 pmol/l. The concentration of CCK in 47 normal fasting subjects ranged from undetectable to 22 pmol/l. Ingestion of a mixed meal in 9 normal subjects increased the plasma concentration from 8.3 +/- 2.5 S.E. to 24.4 +/- 6.5 pmol/l.     

Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and C-natriuretic prohormone peptides compared as early heart failure indicators

OBJECTIVES:The present investigation was designed to determine the best endogenous plasma marker of early congestive heart failure (CHF). METHODS: Forty volunteers with mild CHF (New York Heart Association Class I, n = 12), moderate (Class II, n = 8), or severe (Class III and Class IV, each = n of 5) and 10 age-matched healthy individuals had the simultaneous evaluation of their respective plasma samples by the following radioimmunoassays: atrial natriuretic peptide, ANP; three N-terminal ANP prohormone assays, i.e., proANPs 1-30, 31-67, and 79-98 with the numbers referring to their amino acid (a.a.) sequences in their 126 a.a. prohormone; brain (BNP) and C-natriuretic peptides; N-terminal BNP prohormone; adrenomedullin; neuropeptide Y and endothelin. RESULTS: ProANPs 31-67, 1-30 and 79-98 had 100% (P = 0.01), 83% (P = 0.09) and 50% (P = 0.74) sensitivity in differentiating Class I CHF subjects from healthy subjects. The ANP, BNP, NT-proBNP, CNP, adrenomedullin, neuropeptide Y, and endothelin assays could not differentiate mild CHF subjects from healthy individuals. Logistic regression analysis revealed that only proANP 31-67 significantly (P = 0.0001) discriminated between early CHF (5226 +/- 377 pg/ml) and healthy individuals (1595 +/- 157 pg/ml). The positive and negative predicative values of proANP 31-67 were excellent (100% for each). The peptides measured in these assays were found to be independent markers of CHF with respect to left ventricular ejection fraction. CONCLUSIONS: ProANPs 31-67 is the most sensitive marker in discriminating NYHA Class I CHF subjects from healthy individuals. The ANP, BNP, NT-proBNP, CNP, adrenomedullin, neuropeptide Y and endothelin radioimmunoassays cannot discern mild CHF. These peptides are independent of left ventricular ejection fraction.     

Analytical and clinical evaluation of an electrochemiluminescence immunoassay for the determination of CA 125

The CA 125 II assay on the Elecsys(R) 2010 analyzer was evaluated in an international multicenter trial. Imprecision studies yielded within-run CVs of 0.8-3.3% and between-day CVs of 2.4-10.9%; CVs for total imprecision in the manufacturer's laboratory were 2.4-7.8%. The linear range of the assay extended to at least 4500 kilounits/L (three decades). Interference from triglycerides (10.3 mmol/L), bilirubin (850 micromol/L), hemoglobin (1.1 mmol/L), anticoagulants (plasma), and several widely used drugs was undetectable. Method comparisons with five other CA 125 II assays showed good correlation but differences in standardization. A 95th percentile cutoff value of 35 kilounits/L was calculated from values measured in 593 apparently healthy (pre- and postmenopausal) women. In 95% of patients with benign gynecological diseases CA 125 was 190 kilounits/L. A comparison of CA 125 values obtained with the Elecsys test and with other common CA 125 tests in monitored patients being treated for ovarian cancer showed identical patterns. In conclusion, the Elecsys CA 125 II assay is linear over a broad range, yields precise and accurate results, is free from interferences, and compares well with other assays.     

First direct assay for intact human proinsulin

We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 microL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2-100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1-6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2-18 pmol/L) and 153 pmol/L (98-320 pmol/L), respectively.     

Urinary and plasma urodilatin measured by a direct RIA using a highly specific antiserum

Urodilatin (95-126) (URO) appears to play a major physiologic role in fluid homeostasis and produces major changes when administered intravenously. Here we describe a monospecific, high-affinity antiserum against URO with no cross-reactivity (<0.01%) against the structural highly homologous atrial natriuretic peptide 99-126 (ANP-99-126), ANP analogs, and related peptides such as brain natriuretic peptide. A competitive RIA was developed, based on this antiserum. Urine samples with or without ethanol extraction and plasma samples without pretreatment were analyzed by the RIA, which had a detection limit of 10.5 ng/L, a linear measuring range between 10.5 and 1000 ng/L, and recoveries of 93-102% in urine and 90-104% in plasma. The intraassay CVs were 8.2% and 8.1% for urine samples with 269 and 669 ng/L URO; the interassay CV was 9.7% at 839 ng/L. Using this assay, we present URO data for urine from healthy volunteers receiving low and routine sodium diets and from clinical urine specimens; we also present pharmacokinetic data for URO in plasma from patients suffering from bronchial asthma and treated by URO infusion.     

Glucose availability and the electrophysiology of the human visual system

Immunoreactive parathyroid hormone-related protein (PTH-rP) was measured in simultaneously obtained cerebrospinal fluid (CSF) and plasma from 51 patients suspected of suffering from a prolapsed intervertebral disc. Endocrine or psychiatric diseases were excluded. In addition, immunoreactive parathyroid hormone (PTH) in the CSF samples was measured. Both, PTH-rP and PTH were assayed by immunoradiometric assay. The results indicate the presence of both, PTH-rP and PTH in CSF. The following concentrations (mean values +/- SD) were found: PTH-rP (pmol/l) in CSF without pleocytosis (n = 17) 0.432 +/- 0.157, with pleocytosis (n = 34) 0.654 +/- 0.675; in plasma (pmol/l) 54.1 +/- 14.632; PTH (nmol/l) in CSF without pleocytosis (n = 17) 0.454 +/- 0.099, with pleocytosis (n = 34) 0.437 +/- 0.140, and in plasma 4.272 +/- 0.794. The concentrations of both, PTH-rP and PTH, in CSF with and without pleocytosis were not significantly different. No correlation was found between PTH-rP and PTH values. The present study demonstrated PTH-rP as a normal constituent in human CSF.     

Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer

We have developed a totally automated fluorescence polarization immunoassay for homocyst(e)ine with no pretreatment or chromatographic steps. Comparison with four well-established chromatographic methods yielded r values ranging from 0.980 to 0.997 and slopes from 1.030 to 1.493. Inter- and intraassay CVs ranged from 0.0% to 8.0% and from 0.0% to 6.4%, respectively. Imprecision (CV) of measuring six plasma samples on three instruments ranged from 6.3% to 10.2%. The assay was linear for plasma samples diluted with buffer from 0 to 8-fold. Mean recovery of homocysteine added to two plasma samples was 97.1% and 99.9%. The assay exhibited almost no cross-reactivity towards cysteine and methionine, and a batch of 20 samples can be processed in 60 min.     

Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase II is a thermostable M(r) 540,000 homohexameric alpha-glucosidase with both exo-alpha-1,4-glucosidase and oligo-1,6-glucosidase activities

The lethality of acute renal failure exceeds 50% due to multiorgan dysfunction. In such critically ill patients a reduction of thyroid hormone concentrations without clinical symptoms or laboratory evidence of hypothyroidism frequently occurs. Selenium has recently been shown to play a major role in thyroid hormone metabolism. The aim of this study was to investigate the possible influence of selenium on thyroid hormone metabolism in acute renal failure. Changes in thyroid metabolism were related to the severity of multiorgan failure and to the clinical course. Thyroxine (T4), tri-iodothyronine (T3), free-T4, free-T3, thyrotropin (TSH), serum creatinine, and plasma selenium concentrations in 28 patients (mean age 60 +/- 13) with acute renal failure and multiple-organ dysfunction syndrome were determined initially, and every 3 days after hospital admission. The plasma selenium concentration was found to be reduced compared to normal controls (32 +/- 14 vs. 70-120 micrograms/L). T4 (56 +/- 15 nmol/L, normal range 64-148), T3 (1.31 +/- 0.38 nmol/L, normal range 1.42-2.46), free-T3 (3.1 +/- 1.0 pmol/L, normal range 4.7-9.0), and free-T4 (10.8 +/- 4.0 pmol/L, normal range 10.3-25.8) values were low in 50-70% of the patients at the time of presentation. Plasma TSH concentrations were within the normal range (0.59 +/- 0.79 mU/L, normal range 0.25-3.1), and no clinical symptoms of hypothyroidism were observed. T4 concentration was higher in patients who survived acute renal failure compared to nonsurvivors (62 +/- 22 vs. 51 +/- 16 nmol/L, p < 0.05). Plasma selenium concentration was lower in patients with a severe organ dysfunction syndrome (36 +/- 10 vs. 29 +/- 19 micrograms/L) and correlated with the number of organ failures in these patients (r = -0.247, p < 0.05). T4 and free-T4 values paralleled decreasing selenium concentrations (r = 0.35, p < 0.05). Thyroid hormone levels were reduced in patients with acute renal failure without an increase in TSH. An increase in T4 concentrations became apparent during treatment and may be related to a favorable outcome in acute renal failure. Thyroid hormone concentrations paralleled plasma selenium levels, indicating a possible influence of selenium on thyroid function in acute renal failure.     

Acute and chronic neutral endopeptidase inhibition in rats with aortocaval shunt

In heart failure, sodium and water retention develop despite elevated plasma levels of atrial natriuretic peptide. Atrial natriuretic peptide is degraded in part by a neutral endopeptidase. Whether neutral endopeptidase inhibition improves sodium and water excretion in heart failure is unknown. We determined the effect of neutral endopeptidase inhibition on plasma levels of atrial natriuretic peptide and the renal response to acute volume expansion in rats with aortocaval shunts and in sham-operated controls. Acute endopeptidase inhibition with SQ 28,603 (30 mg/kg) elevated atrial natriuretic peptide plasma levels in both shunted rats (523 +/- 54 to 1258 +/- 330 pmol/L, P<.05) and controls (184 +/- 28 to 514 +/- 107 pmol/L, P<.05). Urinary cGMP excretion, which reflects renal action, increased in parallel. However, the diuretic and natriuretic responses to acute volume expansion were enhanced only in control rats and not in shunted rats. In contrast to the acute effects, chronic neutral endopeptidase inhibition with SCH 34826 (30 mg/kg twice daily) in shunted rats did not change atrial natriuretic peptide plasma levels or cGMP excretion. Nevertheless, the diuretic and natriuretic responses to acute volume load were increased by chronic endopeptidase inhibition in shunted rats (1789 +/- 154 to 2674 +/- 577 microL/80 min and 99 +/- 31 to 352 +/- 96 micromol/80 min, respectively; P<.05). Chronic endopeptidase inhibition attenuated the cardiac hypertrophic response to aortocaval shunt without changing arterial blood pressure. Our data show that the renal effects of neutral endopeptidase inhibition are not necessarily dependent on changes in atrial natriuretic peptide plasma levels but instead may be mediated by local inhibition of the neutral endopeptidase in the kidney. In addition, chronic endopeptidase inhibition may attenuate heart failure-induced cardiac hypertrophy independent of hemodynamic effects.     

Prolactin and beta-endorphin responses to hypoglycemia are reduced in well-controlled insulin-dependent diabetes mellitus

Several pituitary hormones, including corticotropin (ACTH), growth hormone (GH), prolactin, and beta-endorphin (but not thyrotropin, follicle-stimulating hormone, or luteinizing hormone), are released in response to hypoglycemia in normal subjects. In patients with insulin-dependent diabetes mellitus (IDDM), the degree of glycemic control is known to alter ACTH and GH responses to hypoglycemia. The current study was performed to examine the effect of glycemic control on prolactin and beta-endorphin responses to hypoglycemia in subjects with IDDM. We performed 3-hour stopped hypoglycemic-hyperinsulinemic clamp studies (12 pmol/kg/min) during which plasma glucose was decreased from 5.0 mmol/L to 2.2 mmol/L in steps of 0.6 mmol/L every 30 minutes in 20 subjects with uncomplicated IDDM (12 males and eight females; age, 26 +/- 2 years; IDDM duration, 10 +/- 1 years; body mass index, 23.6 +/- 0.6 kg/m2) and 10 healthy subjects (five males and five females aged 30 +/- 1 years). The 10 diabetic subjects in good glycemic control (mean hemoglobin A1 [HbA1], 7.5% +/- 0.3%; normal range, 5.4% to 7.4%) were compared with the 10 poorly controlled patients (mean HbA1, 12.6% +/- 0.5%; P < .001 v well-controlled diabetic group). During hypoglycemia, prolactin levels in the well-controlled diabetic group did not change (7 +/- 1 microgram/L at plasma glucose 5.0 mmol/L to 9 +/- 2 micrograms/L at plasma glucose 2.2 mmol/L), whereas prolactin levels increased markedly in the poorly controlled diabetic group (7 +/- 2 micrograms/L to 44 +/- 17 micrograms/L) and healthy volunteers (12 +/- 2 micrograms/L to 60 +/- 19 micrograms/L, P < .05 between IDDM groups). The plasma glucose threshold required for stimulation of prolactin secretion was 2.2 +/- 0.1 mmol/L in well-controlled IDDM, 3.0 +/- 0.4 mmol/L in poorly controlled IDDM, and 2.4 +/- 0.1 mmol/L in healthy subjects (P < .05 between IDDM groups). Responses in males and females were similar. The increase in beta-endorphin levels was also attenuated in well-controlled IDDM patients (4 +/- 1 pmol/L at plasma glucose 5.0 mmol/L to 11 +/- 4 pmol/L at plasma glucose 2.2 mmol/L) versus poorly controlled IDDM patients (5 +/- 1 pmol/L to 26 +/- 7 pmol/L) and healthy subjects (8 +/- 1 pmol/L to 56 +/- 13 pmol/L). The plasma glucose threshold required for stimulation of beta-endorphin release was again lower in well-controlled IDDM versus poorly controlled IDDM patients (2.2 +/- 0.1 v 3.0 +/- 0.3 mmol/L) and healthy subjects (2.5 +/- 0.4 mmol/L, P < .05 between IDDM groups). In conclusion, prolactin and beta-endorphin responses to a standardized hypoglycemic stimulus (plasma glucose, 2.2 mmol/L) are reduced and plasma glucose levels required to stimulate release of prolactin and beta-endorphin are lower in well-controlled IDDM compared with poorly controlled IDDM and healthy subjects. Thus, stress hormones not previously considered to have a primary role in plasma glucose recovery from hypoglycemia are affected by glycemic control, suggesting a more generalized alteration of hypothalamic-pituitary responses to hypoglycemia in IDDM patients with strict glycemic control.     

Radioimmunossay of secretin in human plasma

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

Ankylosing gout. Apropos of 2 cases

The Miles assay for vascular permeability has high intra- and interassay coefficients of variation (CVs). Quantification, usually by dye extraction and spectrophotometry, is time consuming. In this study, quantification by this means was compared with image analysis using the Olympus CUE-2 Image Analyzer (version 4). The test substance was recombinant human vascular permeability factor (rhVPF). The quantification process took approximately 10 min with image analysis. Formamide extraction and spectrophotometry required 1 hr of preparation, 4-6 days of incubation, and 1 hr for filtration and spectrophotometry. Between assay CVs ranged from 0 to 30% for spectrophotometry, but were all < 10% for image analysis. The sensitivity (2SD above the negative control mean) of the image analysis approach was 64 +/- 25 ng/mL, whereas for spectrophotometry it was 65 +/- 29 ng/mL. Interanimal CVs for rhVPF at 200 and 1000 ng/mL were 15% and 26% when assessed by spectrophotometry and 7% and 22%, respectively, by image analysis. The R2 value for the correlation of image analysis with spectrophotometry was 81.4%. Test substances injected close to the spine evoked a greater permeability response than those injected laterally: at 200 ng/mL p = 0.005, at 1000 ng/mL p = 0.1 (unpaired t tests).     

Analytical and clinical performance characteristics of Tandem-MP Ostase, a new immunoassay for serum bone alkaline phosphatase

The performance characteristics of the Tandem-MP Ostase assay, a new microplate immunoassay for bone-specific alkaline phosphatase (bone ALP; EC 3.1.3.1) in human sera, are described. Bone ALP is bound to streptavidin-coated microwells by a single biotinylated anti-bone ALP monoclonal antibody. Antigen is detected by the addition of p-nitrophenyl phosphate. The assay is performed at room temperature in <90 min. Imprecision was 2.3-6.1% with a detection limit of 0.6 microg/L. Method comparison of bone ALP measurements with the Tandem-MP Ostase assay and the mass-based Tandem-R Ostase assay (n = 285) indicated regression statistics of Tandem-MP Ostase = 1.03 Tandem-R Ostase + 0.22 microg/L, S(y/x) = 4.0 microg/L, r = 0.97. Serum bone ALP values in apparently healthy men and in pre- and postmenopausal women were also similar between the two Ostase assay formats. Liver ALP reactivity determined using the slope and heat inactivation methods was similar in both Ostase assays. Liver ALP reactivity ranged from 3 microg/L (heat inactivation) to 6 microg/L (slope method) per 100 U/L of liver ALP activity, whereas bone ALP reactivity was 37 microg/L per 100 U/L of bone ALP activity, indicating a liver ALP relative reactivity of 8.1-16.2%. Similar results were obtained with the Alkphase-B bone ALP immunoassay. The Tandem-MP Ostase bone ALP assay demonstrated increased concentrations of serum bone ALP in conditions where bone metabolism is increased and showed a rapid, temporal decrease in serum bone ALP in Paget disease patients on bisphosphonate therapy. In conclusion, the Tandem-MP Ostase assay for serum bone ALP is a rapid, simple, robust nonisotopic alternative to the Tandem-R Ostase immunoradiometric assay that provides an accurate and sensitive assessment of bone turnover.