Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS.

The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.

Plasma 2-arachidonoylglycerol is a biomarker of age and menopause related insulin resistance and dyslipidemia in lean but not in obese men and women.

OBJECTIVE: The endocannabinoid system hypertonicity features obesity. Excess circulating 2-arachidonoylglycerol was variously associated with obesity-related metabolic impairment; however, unstandardized experimental and analytical settings have clouded its usefulness as a dysmetabolism biomarker. We aimed at assessing the influence of body mass index (BMI), menopause in women, and aging in men on 2-arachidonoylglycerol relationship with metabolic parameters. METHODS: Adult, unmedicated women (premenopausal (preMW): n = 103; menopausal (MW): n = 81) and men (n = 144) were stratified in normal weight (NW; BMI: 18.5-24.9 kg/m2), overweight (OW; BMI: 25.0-29.9 kg/m2), and obese (OB; BMI ≥ 30.0 kg/m2) classes. Anthropometric and metabolic parameters were determined. Plasma 2-arachidonoylglycerol was measured by a validated liquid chromatography-mass spectrometry assay. RESULTS: 2-arachidonoylglycerol level was raised by menopause (P < 0.001) and by obesity in preMW (P < 0.001) and in men (P = 0.019). In the overall cohorts, 2-arachidonoylglycerol displayed BMI-independent relationships with dyslipidemia (preMW, MW and men), insulin resistance (MW and men), and hypertension (men), but not with waist circumference. Within preMW BMI classes, 2-arachidonoylglycerol correlations were found with triglycerides (P = 0.020) and total cholesterol (TC; P = 0.040) in OB women. In MW, 2-arachidonoylglycerol correlation with triglycerides was found in NW (P = 0.001) and OW (P = 0.034), but not in OB class. Moreover, we found 2-arachidonoylglycerol correlations with TC (P = 0.003), glucose (P < 0.001), and HOMA-IR (P = 0.035) specific for NW MW class. In men, 2-arachidonoylglycerol correlated with triglycerides in NW, OW (both P < 0.001), and OB (P = 0.029), with SBP (P = 0.023) and diastolic BP (DBP; P = 0.048) in OB, and with TC (P < 0.001) in OW class. In NW class 2-arachidonoylglycerol correlations were found with insulin (P = 0.003) and HOMA-IR (P = 0.001), both enhanced by aging (both P = 0.004), and with glucose (P = 0.015) and HDL (P = 0.004). CONCLUSIONS: Plasma 2AG is a biomarker of clustering metabolic dysfunctions, especially in lean men and menopausal women, and could be of help in identifying subjects with elevated cardiometabolic risk despite a healthy anthropometric appearance.

Aldosterone and type 2 diabetes mellitus.

Primary hyperaldosteronism (PA) has recently been demonstrated to be strictly associated to metabolic syndrome as compared with essential hypertension (EH). Besides, the characteristics of metabolic syndrome are different in PA compared to EH, as high fasting glucose is more frequent in the former condition. The adverse effect of excess aldosterone on insulin metabolic signaling has generated increasing interest in the role of hyperaldosteronism in the pathogenesis of insulin resistance and resistant hypertension. Moreover, aldosterone receptor antagonist therapy in diabetic and cardiopathic patients improved coronary flow. The aim of this review is to present recent knowledge about the relationship between aldosterone, insulin resistance and diabetes.

Steroid Profiling by LC-MS/MS in Nonsecreting and Subclinical Cortisol-Secreting Adrenocortical Adenomas.

CONTEXT: Long-term follow-up studies revealed that patients with subclinical hypercortisolism (SH) due to adrenocortical adenomas have an increased incidence of cardiovascular diseases and mortality. No studies have yet investigated the steroid profile and its implications in patients with SH. OBJECTIVE: The objective of the study was to analyze the steroid profile by liquid chromatography-tandem mass spectrometry in sera from patients with unilateral adrenocortical adenomas. DESIGN: This was a cross-sectional study. SETTING: The study was conducted at an outpatient clinic. PARTICIPANTS: Patients with adrenocortical adenomas (nonsecreting, n = 66; SH, n = 28) and 188 age- and sex-matched controls drawn from the general population participated in the study. MAIN OUTCOME MEASURES: Cortisol, 21-deoxycortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, T, progesterone, 11-deoxycorticosterone, and corticosterone in the basal condition and after a 1-24 ACTH test, and clinical data were measured. RESULTS: Patients with SH showed lower basal and 1-24 ACTH-stimulated levels of dehydroepiandrosterone and androstenedione than those with nonsecreting adenomas and controls. T was also lower in SH females. Receiver-operating characteristic curves showed that androgens had good accuracy in predicting SH (sensitivity and specificity were 71% and 76% for dehydroepiandrosterone and 69% and 61% for androstenedione, respectively). Increased cortisol and reduced dehydroepiandrosterone levels were independently associated with increased waist circumference. Cortisol was also independently associated with increased number of cardiovascular risk factors in SH patients. After 1-24 ACTH stimulation, the SH patients also showed increased production of 21-deoxycortisol and 11-deoxycorticosterone. CONCLUSIONS: Liquid chromatography-tandem mass spectrometry steroid profile performed for the first time in sera from patients with adrenocortical adenomas showed impaired secretion of several steroids in SH patients. This fingerprint can help in better characterizing the functional status of these tumors.

Cardiovascular events and mortality in patients with adrenal incidentalomas that are either non-secreting or associated with intermediate phenotype or subclinical Cushing's syndrome: a 15-year retrospective study.

BACKGROUND: Incidental discovery of adrenal masses has increased over the past few years. Mild alterations in cortisol secretion without clinical signs of overt hypercortisolism (subclinical Cushing's syndrome) are a common finding in patients with these tumours. Although metabolic alterations and increased cardiovascular risk have been noted in patients with subclinical Cushing's syndrome, incidence of cardiovascular events and mortality in the long term have not been assessed. We aimed to ascertain the frequency of new cardiovascular events and mortality in patients with non-secreting adrenal incidentalomas, tumours of intermediate phenotype, or those causing subclinical Cushing's syndrome. METHODS: From January, 1995, to September, 2010, consecutive outpatients with adrenal incidentalomas who were referred to the endocrinology unit of S Orsola-Malpighi Hospital, Bologna, Italy, were enrolled into our study. Individuals were assessed every 18-30 months for the first 5 years (mean follow-up 7·5 [SD 3·2] years, range 26 months to 15 years). Cortisol concentrations after the 1 mg dexamethasone suppression test (DST) were used to define non-secreting (+50 nmol/L) and intermediate phenotype (50-138 nmol/L) adrenal incidentalomas and subclinical Cushing's syndrome (+138 nmol/L). At the end of follow-up, patients were reclassified as having either unchanged or worsened secreting patterns from baseline. FINDINGS: 198 outpatients were assessed; at the end of follow-up, 114 patients had stable non-secreting adrenal incidentalomas, 61 had either a stable intermediate phenotype or subclinical Cushing's syndrome, and 23 had a pattern of secretion that had worsened. By comparison with patients with stable non-secreting adrenal incidentalomas, the incidence of cardiovascular events was higher in individuals with a stable intermediate phenotype or subclinical Cushing's syndrome (6·7% vs 16·7%; p=0·04) and in those with worsened secreting patterns (6·7% vs 28·4%; p=0·02). Cardiovascular events were associated independently with a change (from baseline to the end of follow-up) in cortisol concentrations post DST (hazard ratio 1·13, 95% CI 1·05-1·21; p=0·001). Survival rates for all-cause mortality were lower in patients with either stable intermediate phenotype adrenal incidentalomas or subclinical Cushing's syndrome compared with those with stable non-secreting masses (57·0% vs 91·2%; p=0·005). Factors associated with mortality were age (hazard ratio 1·06, 95% CI 1·01-1·12; p=0·03) and mean concentrations of cortisol post DST (1·10, 1·01-1·19; p=0·04). Compared with patients with stable non-secreting adrenal incidentalomas, unadjusted survival for cardiovascular-specific mortality was lower in patients with either a stable intermediate phenotype or subclinical Cushing's syndrome (97·5% vs 78·4%; p=0·02) and in those with worsened secreting patterns (97·5% vs 60·0%; p=0·01). Cancer mortality did not differ between groups. INTERPRETATION: Even when clinical signs of overt hypercortisolism are not present, patients with adrenal incidentalomas and mild hypercortisolism have an increased risk of cardiovascular events and mortality. FUNDING: None.

Cortisol, energy intake, and food frequency in overweight/obese women.

OBJECTIVE: This retrospective study investigated the relation between daily urinary free cortisol excretion rate, as a marker of cortisol production rate, to daily caloric intake, food choice, body mass index (BMI), and waist circumference. METHODS: One hundred twenty-seven overweight/obese women and 21 normal-weight subjects were enrolled in the study. Fasting blood samples for metabolic parameters were taken from each subject, followed by an oral glucose tolerance test. Cortisol excretion rate was assessed on 24-h urine collection (UFC/24 h). In obese patients, the daily caloric intake was calculated, and a weekly food-frequency questionnaire was assessed. Analysis of variance was used to assess the differences between groups. The relations between parameters were investigated by simple and multiple regressions. RESULTS: Obese women had significantly higher UFC/24 h than the normal-weight women (P < 0.001). The obese subjects had an unbalanced diet, particularly rich in saturated lipids, and weekly food choice showed a preference for highly caloric foods. UFC/24 h values and waist circumference were significantly correlated (P < 0.001), regardless of BMI. In the obese group, after adjustment for BMI, the UFC/24 h values were also significantly and positively correlated to daily carbohydrate and lipid intake and to weekly starchy food consumption. CONCLUSION: We demonstrated a significant association between higher UFC/24 h and energy intake, fats, and consumption of starchy foods, and that these relations were independent of BMI.

Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults.

BACKGROUND: The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects. METHODS: 0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis. RESULTS: Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone>1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone<1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established. CONCLUSION: Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.