[Measurement of angiotensin-I-converting enzyme in the blood: help for method validation]. / Dosage de l'enzyme de conversion de l'angiotensine-I sanguine : aide à la validation de méthode.

Blood angiotensin-converting enzyme (ACE) assay is now realized by the determination of enzyme activity on synthetic substrate, mostly furylacryloyl-phenylalanyl-L-glycyl-L-glycine (FAPGG). The matrix can be serum or heparin-plasma, with or without a separator; the assay developed on serum or plasma is not adapted to other matrix such as cerebrospinal fluid where the ACE activity is much lower. This assay has been adapted on a number of automated biochemistry analyzers with the specifications of the supplier of reagents, sometimes with modification of volumes or times for analysis. Samples can be stored at +4̊C for at least for one week, freezing at -20̊C is possible but refreezing is not advised. The assay is linear from 10 to 200 UI/L. Fidelity is excellent after calibration of the assay. Accuracy can be calculated from IQA and EQA results, and the analytical uncertainty is between 2% and 5% in function of the serum ACE value. Usual values will be soon available from studies on age brackets and sex, because ACE activity seems to be more elevated in boys during adolescence. At signature, it is interesting to have medical information on the diagnosis of sarcoidosis or its treatment including ACE inhibitors as a proof of intake; we can give a commentary on elevation of serum ACE activity from other causes than sarcoidosis and the causes for low activities.

Exposure to Glucocorticoids in the First Part of Fetal Life is Associated with Insulin Secretory Defect in Adult Humans.

OBJECTIVE: High glucocorticoid levels in rodents inhibit development of beta cells during fetal life and lead to insulin deficiency in adulthood. To test whether similar phenomena occur in humans, we compared beta-cell function in adults who were exposed to glucocorticoids during the first part of fetal life with that of nonexposed subjects. RESEARCH DESIGN AND METHODS: The study was conducted in 16 adult participants exposed to glucocorticoids during the first part of fetal life and in 16 nonexposed healthy participants with normal glucose tolerance who were matched for age, sex, and body mass index (BMI). Exposed participants had been born to mothers who were treated with dexamethasone 1 to 1.5 mg/day from the sixth gestational week (GW) to prevent genital virilization in children at risk of 21-hydroxylase deficiency. We selected offspring of mothers who stopped dexamethasone before the 18th GW following negative genotyping of the fetus. Insulin and glucagon secretion were measured during an oral glucose tolerance test (OGTT) and graded intravenous (IV) glucose and arginine tests. Insulin sensitivity was measured by hyperinsulinemic-euglycemic-clamp. RESULTS: Age, BMI, and anthropometric characteristics were similar in the 2 groups. Insulinogenic index during OGTT and insulin sensitivity during the clamp were similar in the 2 groups. In exposed subjects, insulin secretion during graded IV glucose infusion and after arginine administration decreased by 17% (P = 0.02) and 22% (P = 0.002), respectively, while glucagon secretion after arginine increased. CONCLUSION: Overexposure to glucocorticoids during the first part of fetal life is associated with lower insulin secretion at adult age, which may lead to abnormal glucose tolerance later in life.

Sex Difference In the Effect of Fetal Exposure to Maternal Diabetes on Insulin Secretion.

We previously showed that fetal exposure to maternal type 1 diabetes (T1D) is associated with altered glucose-stimulated insulin secretion in adult offspring. Here, we investigated whether this ß-cell defect displays a sex dimorphism. Twenty-nine adult nondiabetic offspring of T1D mothers (ODMs) were compared with 29 nondiabetic offspring of T1D fathers. We measured early insulin secretion in response to oral glucose and insulin secretion rate in response to intravenous glucose ramping. Insulin sensitivity and body composition were assessed by a euglycemic, hyperinsulinemic clamp and dual-energy X-ray absorptiometry, respectively. In response to oral glucose, male and female ODMs displayed a reduced insulin secretion. In contrast, in response to graded intravenous glucose infusion, only female ODMs (not males) exhibited decreased insulin secretion. There was no defect in response to combined intravenous arginine and glucose, suggesting that male and female ODMs exhibit a functional ß-cell defect rather than a reduced ß-cell mass. In conclusion, fetal exposure to maternal diabetes predisposes to ß-cell dysfunction in adult male and female offspring. This ß-cell defect is characterized by a sexual dimorphism following intravenous glucose stimulation.

Zinc transporter 8 autoantibodies assessment in daily practice.

OBJECTIVES: Zinc transporter 8 (ZnT8) is specifically expressed in the pancreatic ß-cell and is more restricted in its tissue distribution than other auto-antigens as glutamic acid decarboxylase 65 (GAD65) and insulinoma-associated antigen-2 (IA2). ZnT8 autoantibodies (ZnT8A) assessment allows identifying rapid progression to clinical onset of the disease. We evaluated the prevalence of ZnT8A in adults of different ethnic and phenotypic groups and analyzed its potential utility as additional marker of autoimmunity in daily practice. METHODS: ZnT8A, GADA and IA2A were assessed using enzyme-linked immune-sorbent assay (ELISA) in 160 controls and 216 diabetic subjects. 105 were of type 1 diabetes (T1D), 17 had Latent Autoimmune Diabetes of Adults (LADA), 38 were type 2 diabetic (T2D) and 56 had ketosis-prone diabetes (KPD). 82 patients were newly diagnosed cases. RESULTS: ZnT8A were detected in 1% of controls and were not found in any of our 38 T2D subjects or 56 KPD subjects. In contrast, ZnT8A were detected in 18% of LADA subjects and in 38% of T1D subjects. A slight difference of percentage of ZnT8A positivity was found among our T1D ethnic groups. ZnT8A were positive in 41% of patients positive for GADA and 67% of patients positive for IA2A. The percentage of stratification achieved 91% when GADA, IA2A and ZnT8A were assessed simultaneously. CONCLUSIONS: Results obtained for ZnT8A measurement using ELISA were consistent with previous data. Such investigation could improve the risk stratification and would be integrated in our daily practice.

Evaluation of a new automated assay for the measurement of circulating 1,25-dihydroxyvitamin D levels in daily practice.

BACKGROUND AND OBJECTIVE: 1,25-dihydroxyvitamin D [1,25(OH)2D] is the most important vitamin D metabolite in regulating calcium and phosphorus metabolism and bone resorption. It is measured in plasma for diagnosis and management of patients with disorders influencing vitamin D metabolism. The most common methods for 1,25(OH)2D quantification are competitive isotopic I(125) immunoassays. The purpose of this work is to compare the IDS isotopic immunoassay (IDS RIA) and the nonisotopic iSYS immunoassay (IDS iSYS) for 1,25(OH)2D measurement in human serum and in control samples obtained from the Vitamin D External Quality Assessment Scheme (DEQAS). METHODS: 1,25(OH)2D concentrations in 180 serum samples (87 females and 93 males) and in 25 samples from the DEQAS were assayed using IDS RIA and IDS iSYS methods. Both assays were performed after delipidation and immunoextraction steps. Measurement range was 9.0-348.0pmol/L and 15.6-504.0pmol/L for IDS RIA and IDS iSYS assay, respectively. 'Normal adult' reference ranges of 1,25(OH)2D were 43-168pmol/L for IDS RIA and 63-228pmol/L for IDS iSYS. RESULTS: The equation of the Deming regression analysis demonstrated that IDS iSYS tended to give lower 1,25(OH)2D concentrations by a mean of 20% over the tested concentration range when compared to IDS RIA. Both assays would easily meet the DEQAS goal of 80% of survey samples within 30% of the All-Laboratory Trimmed Mean "ALTM". Results of 1,25(OH)2D obtained using IDS iSYS in samples distributed by DEQAS were closely related to those by LC-MS/MS method. CONCLUSIONS: The concentrations of 1,25(OH)2D measured by the IDS iSYS tended to be lower than those of IDS RIA in patients, but quite comparable to those of LC-MS/MS and ALTM in DEQAS samples.

ß- and α-cell dysfunctions in africans with ketosis-prone atypical diabetes during near-normoglycemic remission.

OBJECTIVE: Ketosis-prone atypical diabetes (KPD) is a subtype of diabetes in which the pathophysiology is yet to be unraveled. The aim of this study was to characterize ß- and α-cell functions in Africans with KPD during remission. RESEARCH DESIGN AND METHODS: We characterized ß- and α-cell functions in Africans with KPD during remission. The cohort comprised 15 sub-Saharan Africans who had been insulin-free for a median of 6 months. Patients in remission were in good glycemic control (near-normoglycemic) and compared with 15 nondiabetic control subjects matched for age, sex, ethnicity, and BMI. Plasma insulin, C-peptide, and glucagon concentrations were measured in response to oral and intravenous glucose and to combined intravenous arginine and glucose. Early insulin secretion was measured during a 75-g oral glucose tolerance test. Insulin secretion rate and glucagon were assessed in response to intravenous glucose ramping. RESULTS: Early insulin secretion and maximal insulin secretion rate were lower in patients compared with control participants. In response to combined arginine and glucose stimulation, maximal insulin response was reduced. Glucagon suppression was also decreased in response to oral and intravenous glucose but not in response to arginine and insulin. CONCLUSIONS: Patients with KPD in protracted near-normoglycemic remission have impaired insulin response to oral and intravenous glucose and to arginine, as well as impaired glucagon suppression. Our results suggest that ß- and α-cell dysfunctions both contribute to the pathophysiology of KPD.

Potential utility of high preoperative levels of serum type I collagen markers in postmenopausal women with primary hyperparathyroidism with respect to their short-term variations after parathyroidectomy.

We evaluated short-term changes in serum amino-terminal procollagen propeptide (P1NP) and cross-linked C-terminal telopeptide (betaCTX) of type I collagen after parathyroidectomy (PTX) in 41 postmenopausal women with primary hyperparathyroidism (PHPT). Serum levels of 25-hydroxyvitamin D, intact PTH, calcium, phosphate, albumin, creatinine, P1NP, and betaCTX were measured before and 2 days after PTX. Their P1NP and betaCTX levels were compared with those measured in 41 normally menstruating and 30 postmenopausal controls. Fifteen of these 41 women had both pre-surgery P1NP and betaCTX concentrations above the upper limit noted in our postmenopausal controls [high turnover (HT) subgroup], while betaCTX levels were solely above the upper limit lastly mentioned in 11 women [high bone resorption (HBR) subgroup]. In addition, these two markers were within the postmenopausal control range in 12 of them [normal turnover (NT) subgroup]. A more significant decrease in postoperative betaCTX levels was observed in the 15 HT compared with the 12 NT PHPT women. The significant postoperative increase in P1NP levels observed in the 15 HT as well as in the 11 HBR was no longer significant in the 12 NT women. In conclusion, higher pre-surgery P1NP and betaCTX levels in post-menopausal PHPT women are associated with a preferential activation of bone formation over bone resorption after PTX.

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?

BACKGROUND: In rainbow trout (Oncorhynchus mykiss), the endocrine control of spermiation is not fully understood. Besides 11ketotestosterone (11KT) and 17alpha, 20beta-dihydroxyprogesterone (MIS), the potential physiological ligand of the mineralocorticoid receptor (MR) 11-deoxycorticosterone (DOC), is a credible candidate in O. mykiss spermiation regulation as spermiation is accompanied with changes in aqueous and ionic flows. METHODS: In this study, we investigated potential roles of DOC during spermiation 1) by describing changes in blood plasma DOC level, MR mRNA abundance during the reproductive cycle and MR localization in the reproductive tract 2) by investigating and comparing the effects of DOC (10 mg/kg) and MIS (5 mg/kg) supplementations on sperm parameters 3) by measuring the in vitro effect of DOC on testis MIS production. RESULTS: The plasma concentration of DOC increased rapidly at the end of the reproductive cycle to reach levels that were 10-50 fold higher in mature males than in immature fish. MR mRNA relative abundance was lower in maturing testes when compared to immature testes, but increased rapidly during the spermiation period, immediately after the plasma rise in DOC. At this stage, immunohistochemistry localized MR protein to cells situated at the periphery of the seminiferous tubules and in the efferent ducts. Neither DOC nor MIS had significant effects on the mean sperm volume, although MIS treatment significantly increased the percentage of males producing milt. However, a significant reduction in the spermatocrit was observed when DOC and MIS were administrated together. Finally, we detected an inhibitory effect of DOC on testis MIS production in vitro. CONCLUSION: These results are in agreement with potential roles of DOC and MR during spermiation and support the hypothesis that DOC and MIS mechanisms of action are linked during this reproductive stage, maybe controlling milt fluidity. They also confirm that in O. mykiss MIS is involved in spermiation induction.

Differences in circulating insulin levels following glargine administration.

OBJECTIVES: To evaluate two-site non competitive insulin immunoassays [BI-INSULIN assay (IRMA) and an electro-chemiluminescent immunoassay (ECLIA)] in routine practice. DESIGN AND METHODS: We studied 145 consecutive patients attending our Endocrine Department for general endocrine explorations and metabolic status investigations. RESULTS: The ECLIA yielded higher results than IRMA in all but six patients treated by glargine. CONCLUSIONS: Insulin levels measured by Elecsys in patients treated by glargine are directly related to glargine biotransformation into detectable metabolite (M1).

Diagnosis of adrenal insufficiency in severe sepsis and septic shock.

RATIONALE: Diagnosis of adrenal insufficiency in critically ill patients has relied on random or cosyntropin-stimulated cortisol levels, and has not been corroborated by a more accurate diagnostic standard. OBJECTIVE: We used the overnight metyrapone stimulation test to investigate the diagnostic value of the standard cosyntropin stimulation test, and the prevalence of sepsis-associated adrenal insufficiency. METHODS: This was an inception cohort study. MEASUREMENTS AND RESULTS: In two consecutive septic cohorts (n = 61 and n = 40), in 44 patients without sepsis and in 32 healthy volunteers, we measured (1) serum cortisol before and after cosyntropin stimulation, albumin, and corticosteroid-binding globulin levels, and (2) serum corticotropin, cortisol, and 11beta-deoxycortisol levels before and after an overnight metyrapone stimulation. Adrenal insufficiency was defined by postmetyrapone serum 11beta-deoxycortisol levels below 7 microg/dl. More patients with sepsis (31/61 [59% of original cohort with sepsis] and 24/40 [60% of validation cohort with sepsis]) met criteria for adrenal insufficiency than patients without sepsis (3/44; 7%) (p < 0.001 for both comparisons). Baseline cortisol (< 10 microg/dl), Delta cortisol (< 9 microg/dl), and free cortisol (< 2 microg/dl) had a positive likelihood ratio equal to infinity, 8.46 (95% confidence interval, 1.19-60.25), and 9.50 (95% confidence interval, 1.05-9.54), respectively. The best predictor of adrenal insufficiency (as defined by metyrapone testing) was baseline cortisol of 10 microg/dl or less or Delta cortisol of less than 9 microg/dl. The best predictors of normal adrenal response were cosyntropin-stimulated cortisol of 44 microg/dl or greater and Delta cortisol of 16.8 microg/dl or greater. CONCLUSIONS: In sepsis, adrenal insufficiency is likely when baseline cortisol levels are less than 10 microg/dl or delta cortisol is less than 9 microg/dl, and unlikely when cosyntropin-stimulated cortisol level is 44 microg/dl or greater or Delta cortisol is 16.8 microg/dl or greater.

Unexpected serum parathyroid hormone profiles in some patients with primary hyperparathyroidism.

BACKGROUND: Third-generation parathyroid hormone (PTH) assays have been reported to measure only intact PTH(1-84), in contrast to second-generation assays, which also detect PTH(7-84) fragments. Higher PTH measurements were observed with third- than with second-generation PTH assays in a few patients with either severe primary hyperparathyroidism or parathyroid carcinoma. METHODS: We analyzed biological data [second- and third-generation PTH assays, 25-hydroxyvitamin D (25-OHD), calcium, and phosphate concentrations] obtained before and after surgery for 2 groups of patients selected from a large series of consecutive patients with primary hyperparathyroidism (PHPT): 7 female patients with surgically and histologically confirmed PHPT (group 1) and a matched group (group 2). RESULTS: For group 1 but not group 2, PTH concentrations measured by third-generation PTH assays before surgery were higher than those measured by the second-generation assays. Circulating 25-OHD, calcium, and phosphate concentrations were similar in both groups. In addition, PTH values measured with the third-generation PTH assays in group 1 decreased after surgery. CONCLUSIONS: Our results confirm that third-generation PTH assays do not measure only PTH(1-84). The frequency of this unexpected finding of markedly lower PTH concentrations than previously reported was approximately 5% in patients with PHPT without malignancy. We do not know whether the presence of this unexpected profile is predictive of malignancy.

Serum bioavailable testosterone: assayed or calculated?

BACKGROUND: Bioavailable testosterone (BT), circulating testosterone not bound to sex hormone-binding globulin (SHBG), is thought to easily penetrate cells. We compared BT measurements obtained by assays with those obtained by calculation with different testosterone association constants. METHODS: We obtained sera from 2 groups of hypogonadal men [group 1 (G1), 1421 samples; group 2 (G2), 170 samples] and a group of healthy men [group 3 (G3), 109 samples]. We added minute doses of [3H]testosterone to the sera, precipitated the SHBG-bound fraction of testosterone with ammonium sulfate (50% saturation), and then assayed serum BT (ABT) as %BT x total. Calculated BT (CBT) was determined with theoretical association constants of testosterone for SHBG (Ks = 1 x 10(9) L/mol) and albumin (Ka = 3.6 x 10(4) L/mol) and paired optimal Ks and Ka values obtained by use of Microsoft Excel software. RESULTS: CBT calculated with theoretical constants differed from ABT by >30% in 85.7% (G1), 84.1% (G2), and 77.9% (G3) of samples, and the mean CBT/ABT ratios were 1.57 (G1), 1.85 (G2), and 1.50 (G3) in spite of fairly good correlations. CBT calculated with paired optimal K(s) and K(a) differed from ABT by <30% in 87.4% (G1), 87.5% (G2), and 97.5% (G3) of samples, and mean CBT/ABT ratios were 0.95-1.04. CONCLUSIONS: To obtain CBT values as close as possible to ABT, optimal paired association constants determined for each studied population must be used instead of the theoretical association constants. Considering the uncertainty of calculating BT, however, use of the ammonium sulfate precipitation method for determining BT is advisable.

Third- or second-generation parathyroid hormone assays: a remaining debate in the diagnosis of primary hyperparathyroidism.

BACKGROUND: Since the demonstration that the second-generation PTH assays, also called intact PTH assays, recognize a non-1-84 PTH fragment in addition to the intact 1-84 PTH, new PTH assays defined as third-generation assays have been commercialized. Two previous studies aimed at evaluating whether these third-generation PTH assays improved the diagnostic sensitivity for primary hyperparathyroidism (PHPT), but they yielded opposite results. METHODS: In the present study we compared two second-generation PTH assays (the total intact PTH assay from Scantibodies Laboratory, Inc., and the intact PTH assay from Nichols Institute Diagnostics) with two third-generation assays (the cyclase-activating PTH assay also from Scantibodies Laboratory and the bio-intact PTH assay from Nichols Institute) in a series of 145 consecutive PHPT patients operated in our endocrine surgery department over a 10-month period. A group of 74 healthy subjects served as controls. RESULTS: The diagnostic sensitivities for PHPT of the total intact, the intact, the cyclase-activating, and the bio-intact assays were 93.8%, 97.3%, 84.2%, and 89.0%, respectively, with 95% confidence intervals in the control groups of 10-46, 11-60, 8.4-34, and 9-41 ng/liter, respectively. CONCLUSION: Our findings demonstrate that the diagnostic sensitivities of second- and third-generation PTH assays are similar. Third-generation PTH assays do not therefore improve the diagnosis of elevated serum PTH levels in PHPT, although there are numerical differences among the values.

Plasma 11beta-hydroxy-4-androstene-3,17-dione: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunoassay using a tritiated tracer.

The plasma concentration of 11beta-hydroxy-4-androstene-3,17-dione (11beta) is very high in 21-hydroxylase deficiency, Cushing's syndrome, and hyperandrogenism of adrenal origin, and very low in congenital 11-hydroxylase deficiency and adrenal insufficiency. Thus, when plasma 4-androstenedione is elevated, it is useful to measure the plasma 11beta level in order to determine the adrenal or ovarian origin of the hyperandrogenism. To eliminate disadvantages related to the 11beta radioimmunoassay (RIA), which uses a tritiated tracer, as well as the high cost associated with scintillation proximity assay (SPA), we developed a non-isotopic 11beta assay that utilizes an 11beta-biotin conjugate synthesized in our laboratory to measure time-resolved fluorescence after addition of streptavidin-europium to microtitration wells. The analytical qualities of this assay are very similar to those of the radioimmunoassay using a tritiated tracer, and an extraction step followed by celite chromatography (which separates 11beta from interfering plasma steroids) prior to a final radioimmuno-competition step. The correlation coefficient between 11beta levels measured by time-resolved plasma 11beta fluoroimmunoassay (TR-FIA) and RIA was 0.965.Finally, the TR-FIA technique was more sensitive and of greater precision than the RIA method.