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NGF and VEGF are known to be involved in different psychiatric diseases. In order to verify hints from basic research that both neurotrophines interact with each other, serum levels of NGF and VEGF were measured in a cohort of 33 healthy individuals and correlated. NGF level was 126.30 pg/mL (±155.43), and VEGF level was 57.28 pg/mL (±44.48). Both factors were significantly correlated, confirming their interaction and legitimising the usage of their respective ratio (0.8 (±0.42)) as a less varying additional marker in prospective studies.

Saliva is gaining increasing attention as a bioanalytical sample matrix. Mostly because of the easy and noninvasive collection, it is not only beneficial in endocrinological and behavioral science, but also in pediatrics. Saliva also has the advantage of being the only body fluid which can be collected even during physical exercise, for example, during sportive activities, and there are physiological characteristics that make it superior to serum/plasma or urine for specific scientific questions. This review provides an insight into the physiology of saliva formation, explaining how certain compounds enter this bodily fluid, and gives advice for collection, storage and analytical methods. Finally, it presents a number of reliable and proven applications for saliva analysis from scientific fields including endocrinology, sports medicine, forensics and immunology.

We examined potential changes in brain-derived neurotrophic factor (BDNF) serum levels and promoter methylation of the BDNF gene in 11 patients with treatment-resistant major depressive disorder during a series of electroconvulsive therapy (ECT). Blood samples were taken before, 1 and 24 h after ECT treatment sessions 1, 4, 7 and 10. Patients remitting under ECT had significantly lower mean promoter methylation rates, especially concerning the exon I promoter, compared to non-remitters (both p < 0.002). These findings may point to a depression subtype in which ECT is particularly beneficial.

Aims Neurotrophins have been linked to the symptomatology of alcohol dependence. We aimed to investigate a possible association between the methylation of the promoters of both neurotrophins, the serum levels of the cytokines and core symptoms of alcohol dependence as withdrawal severity and anxiety. Methods In this study we investigated a possible association between alterations in the methylation of the BDNF IV/NGF I gene promoter and the cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in 55 male alcohol-dependent patients. Results Mean methylation of the promoter of the BDNF gene was significantly associated with the TNF-α serum levels and the CIWA-score during withdrawal (P < 0.001). Moreover, mean methylation of the NGF I promoter was significantly associated with the IL-6 serum levels and STAI-I score during withdrawal (P < 0.001). Conclusion Our results suggest an association between the epigenetic regulation of both neurotrophins, BDNF and NGF, cytokine release and the symptomatology of alcohol dependence. They imply that changes in the methylation of neurotrophins may contribute to the symptomatology of alcohol dependence by affecting relevant downstream signaling cascades.

Summary Assessing the amount of bioavailable cortisol in saliva with immunoassays and thus sampling an endocrine marker of hypothalamus–pituitary–adrenal axis activity is of major interest in both research and clinical practice. However, absolute cortisol concentrations obtained with different immunoassays (IAs) are barely comparable precluding direct comparison between studies or individuals whenever cortisol analyses were not based on the same IA. The present technical report aims to solve this problem by evaluating the validity of, as well as agreement between the most commonly used immunoassays in psychoneuroendocrinological research (i.e., IBL, DRG, Salimetrics, DSL, and DELFIA) and a reference method (LC–MS/MS) in a sample of 195 saliva specimen covering the whole range of cortisol concentrations in adults. A structural equation modelling framework is applied to decompose systematic assay variance and estimate cortisol reference values, which are adjusted for measurement error and interference of salivary cortisone. Our findings reveal nonlinear relations between IAs and LC–MS/MS, which are discussed in terms of IA cross-reactivity with saliva matrix components. Finally guidelines for converting cortisol concentrations being obtained by these immunoassays into comparable reference values are proposed by providing conversion functions, a conversion table, and an online conversion tool.

Preclinical and clinical studies show associations between testosterone and brain-derived neurotrophic growth factor (BDNF) serum levels. BDNF and testosterone have been independently reported to influence alcohol consumption. Therefore, we aimed to investigate a possible interplay of testosterone and BDNF contributing to alcohol dependence. Regarding possible interplay of testosterone and BDNF and the activity of the hypothalamic pituitary axis (HPA), we included cortisol serum levels in our research. We investigated testosterone and BDNF serum levels in a sample of 99 male alcohol-dependent patients during alcohol withdrawal (day 1, 7, and 14) and compared them to a healthy male control group (n = 17). The testosterone serum levels were significantly (p < 0.001) higher in the patients' group than in the control group and decreased significantly during alcohol withdrawal (p < 0.001). The decrease of testosterone serum levels during alcohol withdrawal (days 1-7) was significantly associated with the BDNF serum levels (day 1: p = 0.008). In a subgroup of patients showing high cortisol serum levels (putatively mirroring high HPA activity), we found a significant association of BDNF and testosterone as well as with alcohol craving measured by the Obsessive and Compulsive Drinking Scale (OCDS). Our data suggest a possible association of BDNF and testosterone serum levels, which may be relevant for the symptomatology of alcohol dependence. Further studies are needed to clarify our results.

While the presence of multiple systemic steroids, amines and peptides in saliva has been reported, other hormones of the circulation do not appear in saliva. Substances present within saliva may be classified in different groups: first, those which passively display blood plasma concentrations and constitute a promising alternative to evaluate certain systemic parameters. Second, molecules which seem to play a more active, regulatory role within the upper gastrointestinal tract. Concerning the latter, a growing awareness, especially with regards to salivary peptides has been established. Up to now, understanding the distinct effects of salivary peptides known so far is in its infancy. Various publications, however, emphasize important effects of their presence. Salivary peptides can influence inflammatory processes and cell proliferation in epithelia of the upper digestive tract. These include transforming growth factors (TGFs), epidermal growth factors (EGFs), vascular endothelial growth factors (VEGFs) as well as amines such as melatonin. Of those, candidate cytokines like interleukin 8, tumor necrosis factors (TNFs) and leptin are involved in neoplastic activities of salivary glands and the oral cavity. The exact mechanisms of action are not yet completely understood, but their presence can be utilized for diagnostic purposes. Salivary gland tumors in patients may, in certain circumstances, be identified by saliva diagnostics. Saliva samples of the concerned patients, for instance, reveal significantly higher leptin concentrations than those of healthy individuals. Numerous studies postulate that, beside single indicators, the establishment of salivary hormone profiles may assist clinicians and researchers in detecting tumors and other pathologies of the oral cavity, including adjacent tissues, with high sensitivity and specificity.

Background The hypothalamic galanin expression has been associated with increased intake of carbohydrates and fats in preclinical studies. The appetite stimulating effect of galanin is thought to underlie the positive association between alcohol consumption and hypothalamic galanin expression observed in preclinical studies. Methods In this pilot study we investigated alterations in galanin serum levels (33 male patients) in alcohol-dependent patients during alcohol withdrawal (days 1, 7 and 14) in comparison to healthy controls (19 male controls). In order to assess the putative association between appetite regulation, galanin serum levels and alcohol consumption we additionally investigated the serum levels of insulin, glucose and triglycerides. Results The galanin serum levels on day 1 of alcohol withdrawal were significantly reduced in the alcohol-dependent patients (T = − 3.302, p = 0.002) and increased significantly from day 1 to day 14 of alcohol withdrawal (F = 6.437, p = 0.002). We found a significant negative association between the galanin serum levels and alcohol craving measured by the Obsessive Compulsive Drinking Scale (OCDS) (r=−0.449, p=0.009) and the obsessive subscale of the OCDS (r = − 0.521, p = 0.002) on day 1 of alcohol withdrawal. There was no association between the galanin serum levels and the parameters of energy homeostasis (triglycerides, cholesterol, insulin, and glucose) investigated. Conclusions Acute alcohol withdrawal was associated with decreased galanin serum levels in this pilot study. There was no association between the galanin serum levels and the parameters of energy homeostasis. Further research of galanin serum levels in active drinkers will be necessary to clarify the putative association between galanin serum levels, appetite regulation and alcohol consumption.

Background: Prolactin serum levels have been described to be elevated during alcohol withdrawal in alcohol-dependent patients and normalize during abstinence. Alterations in prolactin levels may reflect disturbances of dopaminergic neurotransmission which is of crucial importance for alcohol-seeking behavior. Methods: In this longitudinal observational study, we investigated prolactin serum levels in 99 male patients during the first 14 days of alcohol withdrawal and early abstinence and in 43 healthy controls. To assess the severity of alcohol dependence, the extent of withdrawal symptoms, craving, depressive symptoms, and anxiety, we employed a structured interview including psychologic measurements. Results: Prolactin serum levels were elevated during the whole study period in alcohol-dependent patients compared to the healthy control group. Prolactin levels at admission (first day of alcohol withdrawal) were associated with the severity of alcohol withdrawal (CIWA-Ar) and of alcohol dependence (SESA) but not with the other assessed psychologic parameters. Conclusions: The presented findings confirm that prolactin is significantly elevated in alcohol-dependent patients during alcohol withdrawal and early abstinence, not showing a rapid decline after cessation of drinking. The association with the severity of withdrawal and dependence may reflect at least partially the individual alterations in the dopaminergic and glutamatergic pathways.

Preclinical study results suggest that brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) modulate addictive behaviour. Therefore we investigated alterations in BDNF (81 male patients) and GDNF serum levels (52 male patients) in alcohol-dependent patients during alcohol withdrawal (day 1, 7 and 14) in comparison to healthy controls (41 male controls). BDNF serum levels were not significantly altered in alcohol-dependent patients compared to healthy controls (p=0.685). GDNF serum levels were significantly reduced in the alcohol-dependent patients (p0.001). BDNF (p=0.265) and GDNF (p=0.255) serum levels did not change significantly during alcohol withdrawal. BDNF serum levels were significantly negatively associated with alcohol withdrawal severity on day 1 (CIWA-Ar score, p=0.004). GDNF serum levels were significantly negatively associated with individual estimation of alcohol tolerance (SESA-XT score, p=0.028). There was no further association with psychometric dimensions of alcohol withdrawal. In conclusion we found that GDNF serum levels are significantly reduced in alcohol-dependent patients. GDNF serum levels were negatively associated with alcohol tolerance. Moreover BDNF serum levels were found to be associated with withdrawal severity.

Background: Since the discovery of autonomous leptin production in salivary glands, very few studies have reported on the physiological or pathological meaning of this particular cytokine in saliva. The aim of this study was to investigate the expression of leptin and its receptors Ob-Ra and Ob-Rb in parotid salivary gland tumors, with particular regard to a potential use of leptin as a tumor marker. Methods: Parotid tissue samples from healthy individuals (n = 31) and tumor patients (n = 97, including tissue samples from pleomorphic adenomas, adenolymphomas, basal cell adenomas, and diverse carcinomas) were analyzed by use of ApoTome-technique microscopy, immunohistochemistry, immunofluorescence, immunoblotting, and quantitative real-time PCR. Salivary and plasma leptin concentrations were measured by using ELISA. Ultrasound was used to determine tumor size before surgery. Results: In all salivary gland tumors leptin was expressed in much higher amounts than in healthy parotid tissues. The cytokine was not imported from the blood but actively produced by the tumors. Immunoblotting results indicated that leptin was present as oligomers in salivary glands. Furthermore, the examined tumors overexpressed the receptor isoforms Ob-Ra and Ob-Rb. Measured leptin concentrations in mixed saliva samples were significantly increased in patients with parotid tumors [mean (SD) 673 (484) pg/mL in pleomorphic adenomas, 679 (465) pg/mL in adenolymphomas, and 880 (618) pg/mL in carcinomas] vs controls [125 (36) pg/mL] (P < 0.001). Conclusions: This is the first study to show that the analysis of salivary leptin in mixed saliva samples may allow preoperative differentiation between tumor patients and healthy individuals.

Introduction: Appetite and eating behavior are frequently altered in psychiatric patients. The newly discovered gut-derived neuropeptide ghrelin simulates hunger and weight gain. Therefore, it might be involved in appetite regulation during psychiatric disorders. Methods: In 83 depressed, 42 schizophrenic patients and 46 healthy controls plasma ghrelin levels were measured, and the psychometric scores on the Three-Factor Eating Questionnaire (TFEQ) were assessed. Results: Neither ghrelin levels nor TFEQ scores differed between both patient groups and healthy subjects. However, TFEQ subscale 2 (disinhibition) was predicted by BMI-corrected ghrelin levels, while age, sex, smoking, and medication did not show any influence. Discussion: Ghrelin correlates with factors of eating behavior, specifically with subscale 2 on the TFEQ. Ghrelin might be involved in appetite-regulating pathways during psychiatric disorders. However, its influence is not likely to be displayed as a difference between diagnostic groups. Rather, ghrelin is associated with eating behavior in psychiatric patients meaning susceptibility to eating problems.

Background: A role of appetite-regulating peptides like leptin and ghrelin in the neurobiology of alcohol craving has been proposed by several studies. Aim of this analysis was to search for differences regarding an association between these peptides and alcohol craving with respect to different subtypes and beverage consumption patterns in patients with alcohol dependence. Methods: We analyzed a sample of 188 patients at admission for alcohol detoxification regarding leptin and ghrelin (n=117) serum levels. Craving was measured using the Obsessive Compulsive Drinking Scale (OCDS). Patients were classified according to Lesch's typology of alcohol dependence and according to their preferred type of alcoholic beverage (beer, wine, spirits). Results: Using general linear models to analyze a possible interaction between subtypes and leptin/ghrelin levels with respect to craving, we found a significant positive association for leptin in patients of Lesch's types 1 and 2, and in patients consuming beer or wine. Ghrelin levels showed a significant trend regarding an association with craving in patients of Lesch's type 1. In the other subgroups we found no significant results. Conclusions: Our findings show that appetite-regulating peptides may be of special importance regarding alcohol craving in subtypes of patients. This may explicate at least in part previous contradictory findings.

Background: We investigated whether ghrelin is present in human saliva, is produced by salivary glands, and physiologic consequences of these findings. Methods: Expression of ghrelin and specific receptor mRNA was determined by PCR. Proteins were identified by immunoblotting and size-exclusion fast protein liquid chromatography (FPLC) with consecutive RIA. Specific RIAs were used for quantification of salivary total and bioactive ghrelin. Distribution of ghrelin was investigated by immunohistochemistry in cryosections of the salivary glands. The effect of ghrelin on incorporation of 5-bromo-2′-deoxyuridine as a measure of cell proliferation was investigated in primary oral keratinocytes. Results:Ghrelin is produced by the salivary glands. The hormone was identified in saliva and glands by immunoblotting and by FPLC fractionation of saliva. Immunohistochemistry demonstrated ghrelin distribution in the salivary glands. The receptor was also produced by the glands and by oral keratinocytes and was shown to be functional. Comparison of total ghrelin values for healthy individuals (body mass index, 18–27 kg/m2) showed significantly lower concentrations in saliva than in serum (P Conclusions: Ghrelin in saliva is produced and released by salivary glands. The effect of ghrelin on oral cell proliferation adds to the pro-proliferative action of other salivary growth factors.

We investigated the influence of salivary leptin, purified by affinity chromatography, on the proliferation of human oral keratinocytes. Furthermore we determined the time- and dose-dependency of the incubation with salivary leptin on the production of epidermal growth factor (EGF) and keratinocyte growth factor (KGF), which are growth factors relevant to keratinocyte proliferation. The analysis was performed both intra- and extracellularly. The relationship between the three cytokines in cell proliferation was studied by successive blocking with specific antibodies. The incubation of oral keratinocytes with recombinant and native leptin led to a significantly increased, dose-dependent cell proliferation (PPPb (the long form of the leptin receptor) expressed by the keratinocytes is functional. It is conceivable that the leptin-induced proliferation in keratinocytes is mediated by this signalling pathway. This is the first study to show a physiological role of salivary leptin as a growth factor for keratinocyte proliferation in the oral cavity. We could demonstrate its influence on the production of other growth factors essential for this proliferative effect. Based on the findings of our study we assume an important role for salivary leptin in wound healing within the vulnerable oral cavity.

In patients with nephrotic syndrome, severe proteinuria is related to significant leptinuria; serum leptin levels remain unchanged. The goal of this study was to elucidate the role of the soluble leptin receptor (sOB-R) in maintaining serum leptin levels in nephrotic patients. Patients with proteinuria were compared with patients in remission of nephrotic syndrome. In this group proteinuria did not exceed 100 mg/m2 of body surface area per day. The period of remission was at least 6 months and was equal in all patients included. The sOB-R level (mean ± sd) in serum of patients with nephrotic syndrome was significantly higher during proteinuria (61.0 ± 17.8 ng/ml) than those in remission or in control patients (36.7 ± 7.0 ng/ml, 36.6 ± 12.0 ng/ml, respectively, P < 0.0001). The ratio between serum leptin levels and the sOB-R (free leptin index) was significantly lower in the proteinuric group (0.012 ± 0.005 vs. 0.06 ± 0.03 and 0.07 ± 0.03 in remission and control group, respectively) (P < 0.001). Urinary sOB-R excretion was similar in all groups. Our data suggest that the counteracting pathway in case of leptin loss in parallel to severe proteinuria in nephrotic syndrome is the up-regulation of its soluble binding protein in serum, which can keep total serum leptin levels constant.

Very few reference intervals for salivary steroids in children have been established to date (1). Even the manufacturers of salivary steroid assays do not provide sufficient reference data for their products. This lack of information is surprising because the measurement of salivary steroids has been accepted as being noninvasive and stress-free (2)(3). In particular, psychiatric and neuro-endocrinologic experiments are frequently designed with saliva as the medium of choice for steroid analysis (4)(5)(6). A large variety of stressors can rapidly affect the adrenal cortex, causing increased adrenal steroid concentrations. For example, hypoglycemia (7) or physical exercise (8) are potent physiologic stressors, whereas fear (9), feelings of inferiority (10)(11), or experiences at school (12)(13) can affect the adrenal cortex activity as psychologic stressors. The taking of blood can also influence adrenal steroid concentrations in children; saliva collection, however, is almost stress-free (14). The use of saliva for steroid analysis in children is therefore an excellent alternative to blood. The aim of our study was to establish age-dependent reference values for salivary cortisol, 17α-hydroxyprogesterone (17OHP), and progesterone in a large cohort of healthy children. The availability of such reference intervals will improve the applicability of saliva analysis as a diagnostic tool in pediatric endocrinology. We collected 252 saliva profiles from healthy children and adolescents (125 boys; age range, 4 days to 15 years; 127 girls; age range, 6 days to 13 years) with normal body length/height and weight. None of the girls had developed a regular menstrual cycle. The parents of the children gave informed consent. Saliva was collected either with the Salivette®, using polyester swabs (Sarstedt), from children >1 year of age or with modified medical pacifiers (Buttner-Frank; see Fig. 1⇓ in the Data Supplement that accompanies the …

Aufgrund der zunehmenden Verwendung von jodiertem Speisesalz fur die industrielle und gewerbliche Lebensmittelproduktion sowie der steigenden Verwendung von Jodsalz in den Privathaushalten sollte mit einer zunehmenden Verbesserung der alimentaren Jodversorgung zu rechnen sein. Bis jetzt waren allerdings grosere regionale Unterschiede zu beobachten. Im Rahmen der FIT-Studie (FIT: family intervention trial) Erlangen, einer Querschnittsuntersuchung des Ernahrungs- und Gesundheitszustands von Schulanfangern und ihren Eltern, wurde deshalb die aktuelle Jodversorgung untersucht, um diese Tendenz auch in dieser Region zu uberprufen. Im Schuljahr 1997/1998 wurden bei Schulanfangern im Alter von 6 und 7 Jahren (n=168) an 16 Erlanger Grundschulen sowie eines Teils ihrer Familienangehorigen (n=251) die Jodausscheidung im Urin sowie die TSH- und fT4-Werte im Serum untersucht. Der Medianwert der Jodkonzentration im Urin aller Schulerinnen und Schuler betrug 11,1 µg/dl bzw. 106,3 µg Jod/g Kreatinin. Mit zunehmendem Alter nahm die Jodausscheidung ab. Der Median fur die Erwachsenen lag bei 8,3 µg/dl bzw. 68,0 µg Jod/g Kreatinin. Wahrend bei den Kindern 53% uber dem WHO-Grenzwert von 100 µg/g Kreatinin lagen, waren es bei den Erwachsenen nur 22%. Etwa 2,8% der Schulanfanger sowie 0,8% der Eltern zeigten eine latente Hypothyreose. Zwischen der Jodausscheidung im Urin und erhohten Serum-TSH-Werten (>4,0 µU/ml) bestand eine signifikante inverse Korrelation (r=–0,83, p=0,005). Die Jodversorgung in der untersuchten Stichprobe in der Region Erlangen hat sich gegenuber den 1980er Jahren verbessert. Dennoch kann sie noch nicht als zufrieden stellend eingestuft werden. Die Jodurinkonzentration muss bei etwa 45% der Schulanfanger und bei etwa 80% der Eltern weiterhin als unzureichend bewertet werden. Die Anstrengungen, die Jodversorgung weiter zu verbessern, durfen nicht nachlassen.

We have analyzed the kinetics of salivary cortisol (F) and 17-hydoxyprogesterone (17OHP) after a single oral administration of hydrocortisone (HC; 10 mg; 0700 h) in healthy male volunteers (n 10; 18 –29 yr) and in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (males, n 7; females, n 3; 8.5–20.4 yr). The HC doses, related to body surface area, ranged from 6.3–9.2 mg/m 2 in controls and from 4.2–10.7 mg/m 2 in CAH patients. Saliva was collected over 5 h (at intervals of 15–30 min), and the steroids were measured with adapted RIAs. In healthy controls, maximal cortisol values (250.3 35.9 nmol/liter) were reached after 30 min. Values showed a monophasic decrease. A t1/2 of 94.5 min was calculated. The proportion of the HC dose in the total area under the curve was 71.2 3.2%. For 17OHP, a monophasic decrease was found, with a minimum level of 48 27 pmol/liter after 300 min. In CAH patients the salivary steroid profiles showed individual kinetics (maximal cortisol values ranged from 107–726 nmol/liter). Here a monophasic decrease was found with a shorter t1/2 of 56.4 min. The HC dose proportion in the area under the curve was 88.3 6%. 17OHP showed biphasic courses with a decrease to the minimum 17OHP level after 210 min at the latest and a subsequent gradual increase. Our findings of limited normalization of the adrenal cortex by oral HC administration underlines the necessity of optimizing therapy control and indicates the usefulness of kinetic studies for the judgement of therapy in CAH patients. (J Clin Endocrinol Metab 87: 1200 –1204, 2002)

We investigated the Cytosin-phosphatidyl-Guanin (CpG) island promoter methylation (mean and methylation of individual CpG-sites) of the nerve growth factor (NGF) gene in the blood of alcohol-dependent patients (57 male patients) during withdrawal (days 1, 7 and 14). Methylation and NGF serum levels did not change significantly from days 1–7. From days 7–14, mean methylation increased (F = 30.55, P

OBJECTIVE: In humans, short term changes of serum leptin lead to alterations in food intake and energy expenditure. The objective of the present study was to relate urine leptin concentrations with the extent of proteinuria in patients with nephrotic syndrome (NS). A second goal was to investigate the impact of potential urinary leptin losses on serum leptin concentrations and body composition. DESIGN AND METHODS: Seventeen patients with proteinuria were compared with twenty patients with remission of NS and ten healthy children. Leptin was measured by radioimmunoassay. RESULTS: Urinary leptin excretion in proteinuric patients was significantly higher than in non-proteinuric patients with and without NS and in healthy controls (2.64+/-0.034 microg/g creatinine, 0.026+/-0.05 microg/g creatinine, and 0.073+/-0.11 microg/g creatinine respectively; P

We studied influences of dental care, food and storage on the reproducibility of salivary steroid levels. Cortisol (F), 17OH-progesterone (17OHP) and Progesterone (P) were measured using adapted commercial radioimmunoassays. Saliva samples of healthy adults (n = 15; m:8; f:7) were collected directly before and after dental care, and directly before and after breakfast with various foodstuffs. A second experiment investigated stability of steroids under different storage conditions. Four series of identical saliva portions (I: Native saliva; II: Centrifuged saliva; III: Saliva with trifluor acetate (TFA); IV: Saliva with 0.5% NaN(3)) were stored at room temperature and at 4 degrees C for up to three weeks. To demonstrate influences of repeated thawing and re-freezing of saliva on steroid values, saliva samples (n = 15) were divided into identical portions. These portions were frozen and re-thawed up to 5 times before measurement. Neither dental care nor intake of bread or milk effected the reproducibility of F, 170HP, and P. Steroid levels decreased significantly in the course of three weeks under different storage conditions (P < 0.001). This decrease was clinically relevant from the second week onward, with exception of NaN(3) treated samples. After repeated freezing and re-thawing 17OHP and P decreased slightly (about 5%). Only F decreased significantly after the third thawing (P < 0.001). The results show the usefulness of standardized handling of saliva samples for improving reproducibility and reliability of salivary steroid measurements.

OBJECTIVE: The objective of the present study was to examine the impact of major heart surgery with cardiopulmonary bypass (CPB) in childhood on serum leptin concentrations in relation to plasma cortisol, epinephrine, norepinephrine, and insulin. DESIGN: Controlled, prospective study. SETTING: Intensive care unit of a university hospital. Patients and INTERVENTIONS: We enrolled 20 pediatric patients undergoing open heart surgery and 20 children with major surgery not necessitating CPB (surgical control group). Leptin was measured by radioimmunoassay, cortisol and insulin were measured by chemiluminescence, and epinephrine and norepinephrine were measured by high-pressure liquid chromatography. MEASUREMENTS AND MAIN RESULTS: In the CPB group, leptin dropped from 0.4 +/- 0.1 preoperatively (mean +/- sem) to 0.2 +/- 0.1 ng/mL intraoperatively (p

Objective : Development of the mucosal immune system is essential for controlling antigenic response. External factors are known to influence the immune system, such as breast-feeding or the mode of delivery. The aim of the present study was to investigate maturation of the enteric immune system. Patients and methods : In stool samples of 59 preterm and term-born infants we measured the concentration of human beta-defensin 2 (HBD 2), an endogenous antimicrobial peptide, and tumor necrosis factor-alpha (TNF-alpha), a cytokine playing a central role in mucosal inflammation, by enzyme-linked immunosorbent assay. Results : Mode of delivery as well as nutrition (breast-feeding or formula) had no influence on the fecal concentration of HBD-2 or TNF-alpha, but there was a significant increase in the concentration of HBD-2 in correlation with gestational age. TNF-alpha showed no change in concentration. Conclusions : Low fecal HBD-2 may be a risk factor in preterm infants to develop neonatal enteric disease, such as necrotizing enterocolitis.

Vascular endothelial growth factor A (VEGF-A) is a key regulator of angiogenesis. This study investigated VEGF-A serum levels during alcohol withdrawal (days 1, 7 and 14, 76 male patients, 38 healthy controls). Patients showed significantly higher VEGF-A serum levels (t = 2.620, P = 0.010), which increased significantly during withdrawal (F = 4.484, P = 0.014, mean difference = -36.835, P = 0.037). The increase of VEGF-A serum levels was significantly associated with initial breath alcohol concentration and the sumscore of the severity scale of alcohol dependence (SESA questionnaire, F = 5.252, P = 0.008). Increase of VEGF-A serum levels is closely associated to alcohol intoxication and severity of alcohol dependence.

Rationale Alterations in the expression of neurotrophic growth factors like brain-derived neuronal factor (BDNF) and glial cell line-derived neuronal growth factor (GDNF) have been frequently associated with affective disorders. Indeed, benzodiazepine dependence is typically combined with affective disorders. Aim of the study was to investigate plasma levels of the neuronal growth factors BDNF and GDNF during benzodiazepine withdrawal in patients suffering from comorbidity of benzodiazepine dependence and depressive disorder.

Disturbances of volume-regulating peptides like vasopressin (AVP) and atrial natriuretic peptide (ANP) have been described in early abstinent alcohol-dependent patients. In a longitudinal approach, we investigated whether changes in AVP and ANP serum levels correlated to cytosine-phosphatidyl-guanine (CpG) methylation of the respective gene promoters on days 1, 7 and 14 of alcohol withdrawal. We analyzed the blood samples of 99 patients suffering from alcohol dependence alongside age- and BMI-matched controls. Concerning AVP promoter methylation, we observed an interaction between time of measurement and CpG loci with CpG 2 showing a significant increase in methylation from day 1 to 14. Serum levels of AVP were significantly decreased in the patient group. Compared to healthy controls, promoter-related DNA methylation of the ANP promoter was significantly reduced on days 7 and 14. Moreover, we detected a significant interaction between CpG position and group. In both cases the difference was mainly observed at CpG 1. The present study shows significant changes in the methylation status of individual CpG sites of AVP and ANP. Observing respective alterations of AVP serum protein levels in alcohol-dependent patients during detoxification treatment, we consider methylation as a possible mode of regulation for these proteins during alcohol detoxification.

Preclinical study results suggest that neurotrophic peptides like nerve growth factor (NGF) and vascular endothelial growth factor A (VEGF-A) may be associated with symptoms of addictive behavior like withdrawal symptoms and rewarding effects. We investigated alterations in NGF and VEGF-A serum levels in opiate-dependent patients (25 male patients), who received diamorphine (DAM, heroin) treatment within a structured opiate maintenance program, and compared the results with the NGF and VEGF-A serum levels of healthy controls (23 male controls). NGF and VEGF-A serum levels were assessed before and after DAM administration twice a day (in the morning (16 h after last application – t1) and in the afternoon (7 h after last application – t3)) in order to detect a possible immediate or summative (in the afternoon) heroin effect on these two neuropeptides. Moreover, we investigated possible associations between the serum levels of these neurotrophic growth factors and psychometric dimensions of addictive behavior, e.g. craving, withdrawal, depression. Whereas there was no direct effect of DAM application on the serum levels of both neurotrophic growth factors neither in the morning nor in the afternoon, the NGF serum levels of the patient group were found to be significantly increased at all four time points of investigation compared with the healthy controls. In contrast, VEGF-A serum levels did not differ significantly in the patient and control groups. We found a significant positive association between the NGF serum levels and several items of the short opiate withdrawal scale as well as a negative association between self-reported mood (measured by visual analogue scale) and mood before heroin application (in the morning as in the afternoon). Moreover, we found a significant positive association between the NGF serum levels (t1 and t3) and the self-reported craving for methadone. In contrast, we found a negative association between the VEGF-A serum levels and avoidance, anxiety, suicide intentions of the SCL-90 as well as a positive association between the VEGF-A serum levels and the subscales of the heroin craving questionnaire measuring the rewarding effects of heroin. In conclusion, the results of this pilot study show that there might be an association between symptoms of opiate dependence and withdrawal and serum levels of VEGF-A and NGF.

Preclinical studies suggest that chronic drug abuse profoundly alters stress-responsive systems. The best studied of the stress-responsive systems in humans is the hypothalamic-pituitary-adrenal (HPA) axis. Apart from cortisol, arginine vasopressin peptide (AVP), and atrial natriuretic peptide (ANP) are known to directly impact upon the HPA axis in addictive behavior. We investigated alterations in ANP, AVP and cortisol serum levels in opiate-dependent patients who received diacetylmorphine treatment within a structured opiate maintenance program. ANP serum levels were significantly increased in opiate-dependent patients as compared to healthy controls, whereas AVP and cortisol serum levels were reduced. The ANP, AVP and cortisol serum levels were not significantly associated with the psychometric dimensions of heroin craving. In conclusion, chronic drug abuse profoundly alters stress-responsive systems like the HPA axis. Alterations of AVP, ANP and cortisol appear to constitute an important component in the neurobiology of opiate-dependent patients.

We investigated the Cytosin-phosphatidyl-Guanin (CpG) island promoter methylation (mean and methylation of individual CpG-sites) of the nerve growth factor (NGF) gene in the blood of alcohol-dependent patients (57 male patients) during withdrawal (days 1, 7 and 14). Methylation and NGF serum levels did not change significantly from days 1-7. From days 7-14, mean methylation increased (F = 30.55, P 0.001), whereas the NGF serum levels decreased significantly (days 7-14: F = 17.95, P 0.001). The NGF serum levels were significantly associated with the mean methylation of the investigated CpG-sites (F = 1.55, P 0.001). These results imply an epigenetic regulation of the NGF gene during alcohol withdrawal.

To determine the usefulness of salivary P and 17 alpha-hydroxyprogesterone (17-OHP) for the assessment of ovarian function. In addition, salivary cortisol (F) levels were measured to assess the role of the adrenal cortex throughout the menstrual cycle.Prospective study.Outpatients in hospital for children and adolescents.Thirty young women with regular menstrual cycles.Saliva collection in the early morning from day 1 of menstrual bleeding until next menses.Salivary P, 17-OHP, and F measured by RIAs.During days 1-12 of the follicular phase, P and 17-OHP levels remained unchanged (P: 9-29.3 pg/mL; 17-OHP: 8-31 pg/mL). Thereafter, P increased exponentially from day 13 onward, reaching a plateau (mean +/- SEM, 70.1 +/- 9.0 pg/mL) between day 16 and 20, followed by a constant decrease until end of the cycle. The 17-OHP levels increased between day 14 and 17 (maximum: 45.8 +/- 4.5 pg/mL), decreasing rapidly thereafter. The F levels remained unchanged (follicular: 7.5 +/- 1.1 ng/mL; luteal 7.2 +/- 1.1 ng/mL). There was a significant correlation between P and 17-OHP (r(2) = 0.43; P.001). When calculating ratios of P/F and 17-OHP/F, linear regression yielded a much stronger correlation (r(2) = 0.74; P.001), although F did not show any correlation to P or 17-OHP.Changes in salivary 17-OHP levels throughout the menstrual cycle reflect ovarian but not adrenal function.

Vascular endothelial growth factor A (VEGF-A) is a key regulator of angiogenesis. This study investigated VEGF-A serum levels during alcohol withdrawal (days 1, 7 and 14, 76 male patients, 38 healthy controls). Patients showed significantly higher VEGF-A serum levels (t = 2.620, P = 0.010), which increased significantly during withdrawal (F = 4.484, P = 0.014, mean difference = -36.835, P = 0.037). The increase of VEGF-A serum levels was significantly associated with initial breath alcohol concentration and the sumscore of the severity scale of alcohol dependence (SESA questionnaire, F = 5.252, P = 0.008). Increase of VEGF-A serum levels is closely associated to alcohol intoxication and severity of alcohol dependence.

The assessment of hormones in saliva has gained wide acceptance in clinical endocrinology. To date, there is no hypothesis as to why some hormones can be found in saliva, while others cannot, and whether there is a physiological consequence of this fact. A number of carefully performed studies give examples of important physiological hormonal activity in saliva. Steroids, such as androgens, act as pheromones in olfactory communication of various mammalian species, such as facilitating mating behavior in swine or serving as odor cues for rodent nestlings. Salivary peptide hormones, such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), and amines such as melatonin, are involved in the regulation of inflammatory processes and in the promotion of cell proliferation, and contribute to a rapid wound healing in the oropharyngeal epithelia. Current data provide evidence of the involvement of salivary cytokines, such as interleukin-8 and leptin, in tumorgenesis in the oral cavity and the salivary glands. The tumor tissues express and release significantly more of these cytokines than healthy glands. Consequently, the assessment of salivary hormone profiles may provide promising targets for diagnostic tumor markers.

Adrenomedullin (ADM) promotes epithelial cell proliferation and antimicrobial activity in the gastrointestinal tract. Since ADM is also present in saliva, it was the objective of our study to investigate the role of salivary ADM in the maintenance of oral health. We found mRNA for ADM and the specific receptors CRLR-RAMP2 and CRLR-RAMP3 expressed by the salivary glands and by oral keratinocytes. The hormone was detected in the glandular tissues by western blot, being slightly bigger than the synthetic peptide, indicating a posttranslational modification. ADM was localized using immunohistochemistry and immunofluorescence. Staining specific for ADM was observed near the cell nuclei of the salivary ducts and acini. There was no correlation between ADM from matched saliva and serum of healthy volunteers. The physiological role of salivary ADM in the oral cavity was investigated by incubating buccal keratinocytes with ADM and measurement of the cell proliferation using bromodeoxyuridine (BrDU) assays. There was a significant, dose dependent increase (up to 5-fold; p

Background: Saliva, which offers a noninvasive and stress-free alternative to plasma and serum, is a widely accepted sample source for analysis of steroids and also of certain amines and peptides. In recent years, numerous publications have described the use of salivary hormone analysis in many fields of clinical and basic research.Content: This review provides an overview of the current applications of salivary hormone analysis. A description of the different modes of hormone entry into saliva is followed by a detailed description of analytical methods and approaches for reliable collection of saliva, including several interesting applications in diverse fields including psychiatry, stress research, clinical endocrinology, sports medicine, and veterinary medicine.Summary: Although saliva has not yet become a mainstream sample source for hormone analysis, it has proven to be reliable and, in some cases, even superior to other body fluids. Nevertheless much effort will be required for this approach to receive acceptance over the long term, especially by clinicians. Such effort includes the development of specific and standardized analytical tools, the establishment of defined reference intervals, and implementation of round-robin trials. One major problem, the lack of compliance sometimes seen in outpatient saliva donors, requires strict standardization of both collection and analysis methods to achieve better comparability and assessment of published salivary hormone data.

Recent studies suggested a role of appetite regulating peptides like leptin and ghrelin in alcohol dependence and particularly in the neurobiology of alcohol craving. Aim of the present study was to investigate alterations of the adipocytokines adiponectin and resistin in alcohol-dependent patients. We analyzed a sample of 88 patients at admission for alcohol detoxification and after 1 week of withdrawal treatment in comparison to 89 healthy controls. Adiponectin and resistin serum levels were measured using commercial ELISA kits. The extent of alcohol craving was obtained using the Obsessive Compulsive Drinking Scale (OCDS). Adiponectin and resistin serum levels were significantly elevated in patients with alcohol dependence at both dates (admission and after 1 week of treatment) compared to healthy controls. Adiponectin decreased significantly during the course of withdrawal (T=3.44, p=0.001) while resistin serum levels showed a slight increase (T=-1.83, p=0.071). In a multivariate approach the extent of alcohol craving was significantly associated with adiponectin but not with resistin serum levels in male patients (Beta=-0.255, p=0.025). Results for female patients were not significant. Our findings provide first evidence for an alteration of the adipocytokines adiponectin and resistin during alcohol withdrawal. Furthermore, adiponectin may be involved in the neurobiology of alcohol craving, possibly via its effects on the hypothalamic circuits.

Recent studies show that alcohol dependence is associated with alterations in plasma levels of nerve growth factor (NGF). The aim of this study was to further elucidate reported alterations in NGF plasma levels during alcohol intoxication and withdrawal. Therefore, we assessed NGF plasma levels by enzyme-linked immunosorbent assay (ELISA) on admission (day 0) and day 7 of alcohol withdrawal in male alcohol dependent patients (n=75) in comparison to healthy controls (n=44). We found significant higher (U=1005.0, p

The recovery of steroids, peptides and therapeutic drugs from commercial saliva collection devices was investigated. Saliva, spiked with defined concentrations of the analytes was applied to the Quantisal, three different Salivettes, and the Saliva-Collection-System to investigate effects of volume, exposure time and temperature on the recovery. Additionally, saliva was collected from healthy subjects with the same devices. It was found that glucocorticoids can be measured very well from samples obtained with the synthetic fiber Salivettes and the Quantisal (80-100%). For androgens, the Quantisal and the Saliva-Collection-System reached recoveries >80%. The Quantisal and polyester Salivette achieved best recoveries (>80%) for peptides. The results for the cotton Salivette were extremely poor for melatonin, insulin or IL-8 (

Background: A sensitive method specific for ghrelins is needed for investigations of this gastrointestinal peptide. Our aim was to develop and validate a quantitative mass spectrometry (MS) method to measure ghrelin and desacyl-ghrelin simultaneously. Methods: After deproteinization by precipitation, we performed reversed-phase separation with a rapid 2-column online extraction design coupled to a quadrupole mass spectrometer for electrospray ionization MS detection. Chromatography was performed on a C18 monolithic column, with ammonium acetate buffer/methanol as the mobile phase and a chromatographic run time of 6 min/sample. The 4-fold–charged ions were used for multiple reaction monitoring experiments. Results: The method was linear with injections of 0.01–10 ng. Limits of detection and quantification were 0.02 and 0.07 μg/L for ghrelin, respectively, and 0.03 and 0.35 μg/L for desacyl-ghrelin. Intra- and interday imprecision (CVs) were 9%–4% and 12%–6% at concentrations of 0.33–5.93 μg/L for ghrelin, respectively, and 16%–6% and 15%–8% at concentrations of 1.12–10.02 μg/L for desacyl-ghrelin. The mean (SD) recoveries in plasma of added ghrelin and desacyl-ghrelin were 95.8% (12%) and 101% (1.2%), respectively. Using kinetic modeling, we determined the mean (SD) periods of half-change (t1/2) of ghrelin to be 156 (16) min in EDTA plasma and 49 (1) min in Li-heparin plasma. Bland–Altman analysis showed that the median differences between EIA and liquid chromatography–tandem mass spectrometry (MS/MS) for desacyl-ghrelin were −40% for plasma/serum samples and 85% for cell supernatants and for ghrelin were 6% for enriched plasma samples and 44% for cell supernatants. Conclusion: Our HPLC-MS/MS procedure has excellent selectivity and sufficient limit of quantification to allow the monitoring of concentration–time profiles in biological matrices.

Background. Natriuretic peptides participate in the complex collection of metabolic effects in reaction to chronic ethanol drinking. Brain Natriuretic Peptide (BNP) is a specific member of this family known to be expressed in heart and brain, thereby being a potential target for the individualization of diagnosis and therapy. Having witnessed effects on other natriuretic peptides in a longitudinal cohort of patients undergoing …

Saliva analysis is an accepted non-invasive alternative to plasma in pediatric endocrinology. Although commercial saliva collectors are available, the reliability of these devices for the analysis of salivary hormones has not been proved. We investigated the recovery and linearity of salivary steroids (cortisol, cortisone, 17-hyroxyprogesterone, testosterone, androstenedione) being relevant in endocrine research and therapy control. Pooled saliva was spiked with ascending concentrations of the steroids and applied onto a variety of absorbents, such as the cotton and the polyester (PE) Salivette (Sarstedt), the foam-tip applicator (Whatman) and strips of blood-spot collection paper (Whatman). Analysis was performed by LC-MS/MS. Best results were achieved using the PE Salivette, yielding recoveries (%) of 99.8 (cortisol), 98.7 (cortisone), 91.8 (17OHP), 96.3 (testosterone), 98.9 (androstendione) with a volume recovery of 98+/-1%. Using the blood-spot paper, recoveries (%) were 92.0 (cortisol), 89.1 (cortisone), 72.0 (17OHP), 70.3 (testosterone) and 77.1 (androstendione). The recovery of glucocorticoids was significantly higher compared to androgens (p

Background: The neuropeptides leptin and ghrelin are involved in the appetite regulating network consisting of distinct orexigenic (ghrelin) and anorexigenic (leptin) circuitries. Recently, it has been shown that elevated leptin levels are associated with alcohol craving in patients suffering from alcoholism. Therefore, the aim of the present pilot study was to determine whether the gut-derived peptide ghrelin which increases hunger and food intake is altered and associated with alcohol craving in alcoholic patients Methods: Two types of alcoholic inpatients, group A (active drinker, acutely intoxicated, n= 97) and group B (early abstainer, who had stopped drinking 24-72 hrs before, n= 21) were consecutively included in a prospective study from the first day of hospitalization. Ghrelin plasma levels and craving data were assessed on days 0, 1, 2 and 7(-10) and compared to those of 24 healthy controls Results: At each time-point ghrelin plasma levels of alcoholic patients were significantly increased compared to healthy subjects. Furthermore, early abstainers showed significantly higher ghrelin levels than active drinkers. In the group of active drinkers ghrelin plasma levels were significantly increased at each time point compared to baseline. No correlations were found between ghrelin levels and craving data measured by the visual analogue scale or the Obsessive Compulsive Drinking Scale Conclusions: Ghrelin levels are elevated in alcoholism and seem to further increase during alcohol withdrawal. However, ghrelin levels do not seem to be associated with alcohol craving.

Plasma 17 alpha-hydroxyprogesterone (17-OHP), androstenedione and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/L (r=0.999). Recoveries (S.D.) of 17-OHP, androstenedione and testosterone in plasma were 100% (5), 102% (2) and 92% (4), respectively. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y=1.942 x+0.255 nmol/L (r=0.695; n=97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for testosterone was y=0.963 x+0.035 nmol/L (r=0.955; n=107). Preliminary reference intervals for children were determined as a function of age and sex. The sensitivity and specificity of the LC-MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.