Association between the epidermal growth factor rs4444903 G/G genotype and advanced fibrosis at a young age in chronic hepatitis C

Background

The epidermal growth factor (EGF) rs4444903 A > G polymorphism has been associated with the development of liver cancer, which commonly complicates cirrhosis of viral origin; however, whether this polymorphism might be associated with fibrosis progression in chronic viral hepatitis is unknown. The present study was performed to assess the allelic and genotypic frequencies of the rs4444903 A > G polymorphism in patients with chronic hepatitis C virus HCV infection and to ascertain whether this polymorphism might be an independent predictor of the degree of fibrosis.

Methods

An RFLP-PCR technique was used to genotype 645 patients (211 with cirrhosis); 528 were referred for the diagnosis and treatment of chronic hepatitis C, and 117 were transplanted for HCV-related end stage liver disease. A group of 428 healthy subjects served as a control. All the subjects were of Caucasian ethnicity.

Results

The EGF rs4444903 A > G polymorphism genotype frequencies in HCV chronic infected patients were as follows: A/A = 227 (35.3%), A/G = 328 (50.9%), and G/G = 90 (14.8%). Genotype frequencies were found to differ between patients with an Ishak staging score ? 2 (A/A = 117, A/G = 157, G/G = 34) and patients with a score > 2 (A/A = 110, A/G = 171, G/G = 56, p = 0.038). A highly significant linear relationship between increasing stage scores and EGF genotype was detected in younger patients (A/A: 2.02 ± 0.18, A/G: 2.55 ± 0.17, G/G: 3.00 ± 0.32, p = 0.008). However, no significant association was detected between the stage score and EGF genotype in older patients (A/A: 3.79 ± 0.19, A/G: 3.64 ± 0.15, G/G: 3.98 ± 0.30 p = 0.579).

Conclusions

The EGF rs4444903 A > G polymorphism may facilitate liver fibrosis progression in Caucasian patients with chronic hepatitis C, especially in younger patients.     

Development of anabolic-androgenic steroids purity certified reference materials for anti-doping

Background

The need for certified reference materials (CRM) of anabolic-androgenic steroids reference materials was emphasized by the Beijing 2008 Olympic game as a tool to improve comparability, ensuring accuracy and traceability of analytical results for competing athletes. The China National Institute of Metrology (NIM) responded to the state request by providing seven anabolic-androgenic steroids (AAS) reference materials for Beijing Olympic anti-doping, GBW (E) 100086-GBW (E) 100092.

Experimental

This work describes the production of the series of AAS CRMs, according to ISO Guides 34 and 35 [1] and [2], which comprises the material processing, homogeneity and stability assessment, CRMs’ characterization including moisture content, trace metal content. The AASs’ purity values were assigned with collaborative study involved eight laboratories applying high resolution liquid chromatography-diode array detector (HPLC-DAD). Homogeneity of the AAS CRMs were determined by an in-house validated liquid chromatographic methodology. Potential degradation during storage was also investigated and a shelf-life based on this value was established.

Results

The certified values of CRMs were 99.76 ± 0.079%, 99.76 ± 0.25%, 99.63 ± 0.09%, 99.67 ± 0.11%, 98.82 ± 0.56%, 96.30 ± 0.39% and 99.71 ± 0.49% (purity ± expanded uncertainty with confidence level of 95%) for methyltestosterone, testosterone propionate, nandrolone, nandrolone 17-propionate, boldenone, trenbolone acetate and testosterone respectively.

Conclusions

The certified values for all the studied AAS reference materials are traceable to the international system of units (SI). The CRMs developed were applied by 32 laboratory including sports organizations and analytical laboratories during the 2008 Olympic game for anti-doping control.     

Steroid hormone levels associated with passive and active smoking

Context

Cigarette tobacco smoke is a potent environmental contaminant known to adversely affect health including fertility and pregnancy.

Objective

To examine the associations between second-hand cigarette tobacco-smoke exposure, or active smoking and serum concentrations of steroid hormones using tandem mass spectrometry.

Design

Healthy women (18-45 y) from the general community in the Metropolitan Washington, DC were recruited at the follicular stage of their menstrual cycle. Participants were assigned to one of three study groups: active smokers (N = 107), passive smokers (N = 86), or non-smokers (N = 100). Classifications were based on a combination of self-reporting and serum cotinine concentrations.

Methods

Serum androgens, estrogens, progestins, androstenedione, aldosterone, cortisol, corticosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 11-deoxycortisol and 25-hydroxy-vitamin D3 (25-OHVitD3) and cotinine were measured by isotope dilution tandem mass spectrometry (LC/MS/MS) (API-5000). Kruskal-Wallis tests were used to assess median differences among the three groups, with Dunn's multiple comparison test for post hoc analysis.

Results

Serum estrone, estradiol, and estriol concentrations were lower in active and passive smokers than in non-smokers. The three study groups differed significantly in serum concentrations of 16-OHE1, aldosterone and 25-OHVitD3, as well as in the ratios of many of the steroids. Pair-wise comparison of the groups demonstrated significant differences in hormone concentrations between (i) smokers and non-smokers for aldosterone: (ii) passive smokers and non-smokers for aldosterone, progesterone and estriol. Moreover, for smokers and passive smokers, there were no significant differences in these hormone concentrations.

Conclusions

Smoke exposure was associated with lower than normal median steroid hormone concentrations. These processes may be instrumental in explaining some adverse effects of tobacco smoke on female health and fertility.     

Clinical significance of plasma CD146 and P-selectin in patients with type 2 diabetic nephropathy

Objective

To investigate the levels of plasma CD146 and P-selectin in patients with type 2 diabetic nephropathy at different stages.

Methods

A total of 80 patients with type 2 diabetes mellitus were enrolled in the present study. According to 24 h urinary albumin excretion ratio and renal function, they were further divided into group of diabetes without microalbuminuria (DN0, n = 20), microalbuminuria group (DN1, n = 20), macroalbuminuria group (DN2, n = 20) and renal insufficiency group (DN3, n = 20). Another 20 healthy subjects were enrolled as control group (non-DM). Plasma CD146 and P-selectin were measured by ELISA.

Results

Plasma CD146 and P-selectin were significantly increased in patients with type 2 diabetes with microalbuminuria (DN1) compared with health control (CD146: 415.3 ± 29.0 vs. 243.5 ± 14.7 ng/ml, P < 0.05; P-selectin: 66.8 ± 3.4 vs. 45.3 ± 2.7 ng/ml, P < 0.001). With the development of diabetic nephropathy, both plasma CD146 and P-selectin level progressively rise, with the highest levels in patients with significant renal insufficiency (DN3: 515.9 ± 36.9 and 81.5 ± 5.1 ng/ml respectively, P < 0.001). Moreover, the increase in CD146 is positively co-related to the rise of P-selectin in patients with type 2 diabetes.

Conclusion

Expression of CD146 and P-selectin in patients with type 2 diabetes is elevated, and they are positively correlated with severity of diabetic nephropathy.     

Neurohormonal and cytokine fluctuations following transcatheter closure for an atrial septal defect

Introduction

Inflammation and neurohormonal activation are considered to be involved in the development of earlier and/or later complications in congenital heart disease patients, even after a successful repair of the lesion. It is not yet clarified what is the role of the therapeutic interventions in the occurrence of such a response and how it could be associated with possible postoperative complications.

Aim

We sought to assess the inflammatory and neurohormonal response to transcatheter closure of secundum type atrial septal defects (ASD) over a six-month follow-up period. We also evaluated the association between the respective markers and catheterization data as well as echocardiographic measurements.

Methods

Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), N-terminal-proatrial natriuretic peptide (NT-proANP) and N-terminal-probrain natriuretic peptide (NT-proBNP) were assessed and echocardiographic measurements were performed in twenty-eight patients with atrial septal defect prior to, and at the first, second and sixth months post transcatheter closure. Thirty-three age-matched healthy volunteers were also enrolled.

Results

IL-6 plasma levels, although higher preoperatively, [physical logarithm (ln) IL-6: 3.37 ± 0.66 vs 2.92 ± 0.44 pg/ml, p = 0.015], reached control levels postoperatively, at the end of the third month, whereas TNF-α and IL-10 were not influenced by the procedure. NT-proANP levels were elevated preoperatively compared to the control group (ln NT-proANP 3.78 ± 0.572 vs 3.48 ± 0.30, p = 0.031), with a further significant increase during the 1st month (ln NT-proANP 3.78 ± 0.572 vs 4.2 ± 0.42, p = 0.006), following the pattern of the left atrial volume enlargement, and remained high even 6 months after the procedure .On the other hand, the initially normal concentrations of NT-proBNP, after a transient significant increase during the first month postoperatively (ln NT-proBNP 3.56 ± 0.94 vs 4.58 ± 0.91, p < 0.0001) returned to the controls’ levels at the end of the third month. Preoperative concentrations of NT-proANP positively correlated with NT-proBNP concentrations and pulmonary to systemic flow ratio (Qp/Qs).

Conclusions

Transcatheter closure could improve, on a mid- term basis, the inflammatory process but natriuretic peptides’ secretion continues in parallel with left atrial volume increase. Further follow up is required to determine the long-term progress of the inflammatory and neurohormonal response to the procedure.     

The relative contribution of environmental and genetic factors to phenotypic variation in familial Mediterranean fever (FMF)

Introduction

Familial Mediterranean fever (FMF) is an autosomal recessive disease, caused by mutations in the FMF gene MEFV (MEditerranean FeVer). It has a large phenotypic diversity even in patients with similar genotypes. Despite evidence that environmental factors (EFs) and genetic factors, including MEFV mutations (such as M694V, E148Q) and background modifier genes (MGs), affect the clinical manifestations of FMF, the relative contribution of each remains unknown.

Methods

To investigate the relative contribution of environmental and genetic factors to the phenotype of FMF, we compared the intra-pair clinical concordance of 10 mono and 7 dizygotic twins with FMF. The part played by EFs was determined by the phenotypic discordance of the monozygous twins, and the MGs effect was determined by deducing the environmental effect, computed for MZ twins, from the phenotypic discordance of the dizygous twins.

Results

The mean ± SD of intra-pair concordance was higher in the MZ than in DZ twin group (88.1 ± 13.2 vs. 70.7 ± 14.1 respectively, P value < 0.05). Based on the concordance in clinical manifestations in MZ and DZ twins, the environmental effect on the phenotype of FMF is estimated as 11.9% ± 6.6% and the MGs effect as 17.4% ± 15.5% in average.

Conclusions

In FMF the phenotype is affected by MEFV mutations, MGs and EFs in an estimated ratio of about 6:1.5:1 respectively.     

Liquid chromatography-tandem mass spectrometry analysis of urinary fluticasone propionate-17beta-carboxylic acid for monitoring compliance with inhaled-fluticasone propionate therapy

Background

Inhaled corticosteroids including fluticasone propionate (FP) are the most effective treatment for persistent-asthma. Noncompliance ranging from 20% to 80% of treated patients is associated with substantial health care costs, morbidity and fatalities. A noninvasive test to assess FP treatment compliance is needed. The major metabolite of FP is FP-17beta-carboxylic acid (FP17βCA) and is excreted in urine. This study demonstrates the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure FP17βCA in urine and evaluation of FP17βCA urinary elimination.

Experimental

Fluorometholone was used as the internal standard. After acetonitrile precipitation, samples were extracted with dichloromethane, washed and dried. Reconstituted extract (60 μL) was subjected to reversed-phase chromatography and positive-ion mode LC-MS/MS analysis. Assay precision, linearity, recovery and sample stability were determined. Elimination evaluation included measurement of FP17βCA in urine collected daily from human subjects before (day 1), during treatment (days 2-5; dose FP-110 μg 2 puffs/day), and following cessation of FP therapy (days 6-14; n = 4).

Results

Linear range of the FP17βCA assay was 10.3-9510 pg/mL. Limit of quantitation (LOQ) was 10.3 pg/mL and recovery ranged from 85.8% to 111.9%. Inter-assay CVs were 7.4-12.0% for FP17βCA concentrations of 11.1-5117 pg/mL. Urine FP17βCA was absent in subjects prior to FP therapy, detectable (180-1991 ng FP17βCA/g creatinine) throughout the dosing period and reached below the LOQ at 6 days after therapy cessation.

Conclusions

Measurement of FP17βCA by LC-MS/MS has acceptable analytical performance for clinical use. These data support the clinical utility of measuring FP17βCA in urine to monitor patient compliance with FP therapy.     

Association of TNF-α promoter gene G-308A polymorphism with metabolic syndrome, insulin resistance, serum TNF-α and leptin levels in Indian adult women

Background

Tumour necrosis factor alpha is a multifunctional proinflammatory cytokine involved in the pathogenesis of metabolic syndrome, insulin resistance, and obesity. Aim of this study is to investigate in a North Indian female population the impact of the G-308A TNF-α variant on various components of the metabolic syndrome, Insulin Resistance, serum TNF-α and Leptin levels.

Methods

The G-308A TNF-α polymorphism has been studied in 269 females with metabolic syndrome (NCEP ATP III criteria) (age 31.91 ± 6.05) and 272 healthy females without metabolic syndrome (age 30.96 ± 7.01). The G-308A variant was detected by PCR amplification and Nco-1 digestion.

Results

Homozygous mutant genotype (AA) (p = <0.001: OR = 3.24: 95% CI = 2.15-4.89) and mutant allele (A) (p = <0.001: OR = 3.04: 95% CI = 2.08-4.43) of TNF-α was significantly less frequently observed in the control population as compared to study group. Furthermore, on dividing the subjects into two groups according to the absence (TNF-1 allele) or presence of the mutant A (TNF-2) allele, significant results were obtained in most of the metabolic risk factors.

Conclusions

Our results suggest that the G-308A polymorphism of the TNF-α gene may be independently associated with hypertension, leptin level and hypercholesterolemia leading to metabolic syndrome independent of Insulin resistance and hyperglycemia.     

Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

Objective

To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism.

Methods

Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different static pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure.

Results

Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 ± 2.8 mg/g, 31.8 ± 0.7 mg/g, 92.3 ± 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 ± 9.4 mg/g, 235.9 ± 3.0 mg/g, 386.7 ± 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels were upregulated.

Conclusion

Static pressures stimulate ox-LDL-induced cholesterol accumulation in cultured VSMCs through decreasing caveolin-1 expression via inducing the maturation and nuclear translocation of SREBP-1.     

WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Abolished circadian rhythm of salivary cortisol in elite artistic gymnasts

Objective

The aim of this study was to evaluate the effects of intensive physical exercise and acute psychological stress during high level athletic competition as reflected on the levels of salivary cortisol in elite artistic gymnasts (AGs).

Design

The study included 239 AGs (142 females-97 males) who participated in the European Championship of Gymnastics in 2006 and 81 adolescents (40 females-41 males), matched for age, as controls. All athletes participated voluntarily in all or parts of the study, providing samples or data for each of the variables measured. Height, weight, body fat, lean body mass (LBM), bone age and Tanner stage of puberty were assessed and data concerning the time of thelarche, adrenarche and menarche as well as, the onset and the intensity (hours per week) of training were obtained.

Methods

Saliva samples were collected, the morning before training and in the afternoon shortly after the competition. From controls, the saliva samples were collected in the morning. Cortisol concentrations were measured using a chemiluminescence method. Acute stress was assessed using a questionnaire designed for the study.

Results

No difference was found between morning and afternoon salivary cortisol levels in both male and female AGs (females: AM: 15.45 ± 7.45 nmol/l vs PM: 15.73 ± 9.38 nmol/l; males: AM: 10.21 ± 5.52 nmol/l vs PM: 9.93 ± 13.8 nmol/l, p > 0.05).Female AGs presented higher levels of morning salivary cortisol than female controls (p < 0.05). Both male and female AGs had higher degree of psychological stress in comparison with controls (p < 0.001, p < 0.013, respectively). Female AGs had higher morning and afternoon salivary cortisol levels (p < 0.01, p < 0.01, respectively) and higher degree of stress (p < 0.003) than males.

Conclusions

In elite AGs the diurnal rhythm of salivary cortisol has been abolished, probably due to the strenuous training and competition conditions. Female AGs presented higher levels of morning salivary cortisol and psychological stress compared to both male AGs and female controls. The long term consequences of these modifications of the HPA axis remain to be elucidated.     

Síntomas depresivos en adultos mayores participantes de una universidad abierta a la tercera edad

Introduction

Although depressive disorders prevalence among older adults usually is less than in the younger population, the presence of significant symptoms of depression is common in elderly people. Studies show that taking part in social, educational and pleasure activities is associated with a reduction in depressive symptoms in this population. The purpose of this study was to examine the prevalence of depressive symptoms above the elderly participants of a Third Age Open University, taking the time taking part as a reference.

Material and methods

A cross-sectional design was implemented, with a participation of 95.2% (n = 140) of the total enrolled in the first trimester of 2009 in the activities of the Third Age Open University of the Schools of Arts, Sciences and Humanities of the University of São Paulo. They all answered a socio-demographic questionnaire and the Geriatric Depression Scale (GDS-15).

Results

The prevalence of depressive symptoms found was 3.57%. The statistical analysis showed a relationship between participation formore than one semester in the Third Age Open University and a lower index of depressive symptoms (p < .05).

Conclusions

Participating for longer than one academic semester is associated with less depressive symptoms, possibly being a protector factor against depression.     

Fragilidad y dependencia en Albacete (estudio FRADEA): razonamiento, diseño y metodología

Objective

To obtain a cohort of subjects of equal to or greater than 70 years, representative of a Spanish urban population, to estimate the prevalence of frailty and follow it up over time to analyse associated factors.

Material and methods

A prospective, population-based cohort study. From a population of 18,137 elderly persons, a representative sample of 1172 was randomly stratified, of which 993 (84.7%) agreed to take part. The variables collected were; sociodemographic, comorbidity, functional (n = 825), cognitive, affective and quality of life. On the patients who agreed, body composition was determined by bioimpedance analysis (n = 557), basal metabolic rate by indirect calorimetry (n = 450) and a blood sample was obtained for biomarkers (n = 859). Frailty was defined by the presence of 3 or more Fried criteria: unintentional weight loss, low energy, exhaustion, slow walking, and low physical activity. The cohort will be followed up over time until the death of the subjects.

Results

Mean age 79.4 (SD 6.4) years, with 601 (60.5%) women. A total of 21.3% were institutionalised; 16.9% were frail, 48.5% pre-frail, 21.3% non-frail, and 12.8% did not have the 3 criteria to be able to determine their state, of which 9.5% had moderate-severe incapacity, which would increase the prevalence of frailty to 26.4%.

Conclusions

A FRADEA cohort has been constructed, representative of an urban population in Spain. The prevalence of frailty in the cohort was 16.9%.     

Apport de deux techniques d’analyse automatisées des examens [F] FDG TEP dans la discrimination des patients déments frontotemporaux et malades d’Alzheimer

Introduction

In younger patients, the in vivo clinical diagnosis of Alzheimer's disease (AD) and of the frontotemporal type (FTD) may be cumbersome. The gold standard diagnostic proof is currently still based upon pathology examination. It is crucial to find reliable techniques to make an accurate in vivo diagnosis and to differentiate the etiology of the dementia.

Patients and method

Twenty-four patients bearing clinically diagnosed AD (n = 16) and FTD (n = 8) underwent [¹⁸F] FDG-PET/CT brain scan. Four nuclear medicine physicians with varying expertise in neuroimaging read each scan according to: visual analysis; automated analysis computed by BRASS^® Hermes^® software; automated analysis computed by Cortex ID^® General Electric^® software. Interpretation aimed at assessing the global scan aspect, the cerebral metabolism per hemisphere (in five relevant regions) and the diagnostic degree of confidence. Diagnostic interpretations derived from visual and automated analyses were compared to clinical diagnosis. Inter-observer agreement and Kappa scores were calculated.

Results

Kappa analyses showed a gain in diagnostic accuracy for a nonexpert physician, a gain in diagnostic confidence with Cortex ID^® and a gain in interobserver diagnostic agreement with BRASS^®.

Conclusion

Using automated software such as Cortex ID^® or BRASS^® helps standardizing the interpretation of [¹⁸F] FDG distribution pattern in AD or FTD.     

Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects

Background

α-Klotho (αKl) regulates mineral metabolism such as calcium ion (Ca²⁺) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type αKl has been demonstrated, less is known regarding the physiological importance of soluble-type αKl (sαKl) in circulation.

Objectives

The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum sαKl, and (2) to determine reference values for sαKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects.

Results

We successively developed an ELISA to measure serum sαKl in healthy volunteers (n = 142, males 66) of ages (61.1 ± 18.5 year). The levels (mean ± SD) in these healthy control adults were as follows: total calcium (Ca; 9.46 ± 0.41 mg/dL), Pi (3.63 ± 0.51 mg/dL), blood urea nitrogen (BUN; 15.7 ± 4.3 mg/dL), creatinine (Cre; 0.69 ± 0.14 mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)₂D; 54.8 ± 17.7 pg/mL), intact parathyroid hormone (iPTH; 49.2 ± 20.6 pg/mL), calcitonin (26.0 ± 12.3 pg/mL) and intact fibroblast growth factor (FGF23; 43.8 ± 17.6 pg/mL).Serum levels of sαKl ranged from 239 to 1266 pg/mL (mean ± SD; 562 ± 146 pg/mL) in normal adults. Although sαKl levels were not modified by gender or indices of mineral metabolism, sαKl levels were inversely related to Cre and age. However, sαKl levels in normal children (n = 39, males 23, mean ± SD; 7.1 ± 4.8 years) were significantly higher (mean ± SD; 952 ± 282 pg/mL) than those in adults (mean ± SD; 562 ± 146, P < 0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that sαKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum sαKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of α-klotho gene. In this patient, sαKl level was notably lower than those of age-matched controls.

Conclusion

We established a detection system to measure human serum sαKl for the first time. Age, Ca and Pi seem to influence serum sαKl levels in a normal population. This detection system should be an excellent tool for investigating sαKl functions in mineral metabolism.     

Urinary free cortisone, but not cortisol, is associated with urine volume in severe obesity

Background

High urine volume enhances urinary free cortisol (UFF) and cortisone (UFE) excretion rates in normal-weight adults and children. Renal excretion rates of glucocorticoids (GC) and their metabolites are frequently altered in obesity. The aim of the present study was to investigate whether UFF and UFE excretion is also affected by urine volume in severely obese subjects.

Experimental

In 24-h urine samples of 59 extremely obese subjects (mean BMI 45.3 ± 8.9 kg/m²) and 20 healthy lean subjects (BMI 22.1 ± 1.8 kg/m²), UFF and UFE, tetrahydrocortisol (THF), 5α-tetrahydrocortisol (5α-THF), and tetrahydrocortisone (THE) were quantified by RIA. The sum of THF, 5α-THF, and THE (GC3), the three major GC metabolites, reflects daily cortisol secretion. 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity was assessed by the ratio UFE/UFF. Daily GC excretion rates were corrected for urine creatinine and adjusted for gender and body weight.

Results

In extremely obese subjects, urine volume was significantly associated with creatinine-corrected UFE and 11β-HSD2 activity after adjustment for gender and BMI (r = 0.47, p = 0.0002 and r = 0.31, p = 0.02, respectively). However, urine volume was not associated with creatinine-corrected UFF and GC3 (p = 0.4 and p = 0.6, respectively). In lean controls, urine volume was significantly associated with creatinine-corrected UFE and UFF (r = 0.58, p = 0.01 and r = 0.55, p = 0.02, respectively), whereas urine volume was not associated with 11β-HSD2 activity after appropriate adjustment (p = 0.3).

Conclusions

In severe obesity, in contrast to normal weight, renal excretion of UFE, but not of UFF is affected by fluid intake. This discrepancy may be due to the increased renal 11β-HSD2 activity in obesity.     

A re-investigation of saliva collection procedures that highlights the risk of potential positive interference in cortisol immunoassay

Background

The use of saliva for measurement of cortisol permits non-invasive study of adrenal function, but collection can be technically difficult, particularly in small infants. Saliva collection can be assisted by citric acid to increase saliva flow, or by the use of cotton or polyester swabs in the mouth.

Aim

To determine whether different methods of saliva collection affect cortisol radioimmunoassay (RIA) performance.

Experimental

Cortisol was measured in saliva collected from 16 adults using intra-oral cotton swabs or polyester swabs, compared with saliva dribbled directly into a pot either alone (plain saliva) or after citric acid had been placed on the tongue. An in-house RIA, without prior extraction, was used to measure cortisol with an encapsulated sheep antibody.

Results

Mean (median) salivary cortisol was 10.9 (10.5) nmol L⁻¹ in plain saliva, 10.4 (8.4) nmol L⁻¹ in citric acid stimulated saliva; 25.3 (25.1) nmol L⁻¹ in saliva collected on cotton swabs, and 27.9 (27.3) nmol L⁻¹ collected on polyester swabs. Cortisol in saliva collected using citric acid was not significantly different from plain saliva (p = 0.997), but cortisol in saliva collected using cotton and polyester swabs was significantly higher than that of plain saliva (p < 0.01).

Conclusion

The use of cotton or polyester swabs for collection of saliva can result in spuriously high levels of cortisol when measured by RIA.