Ghrelin plasma concentration increases in parallel to cortisol after a standardized psychological stress in humans, but the physiological basis of this interaction is unknown. We aimed to elucidate this question by studying the ghrelin response to pharmacological manipulation of the hypothalamic–pituitary–adrenal (HPA) axis. Six lean, healthy male volunteers were examined under four experimental conditions. Blood samples were collected every 30 min for two sequential periods of two hours. Initially, a baseline period was followed by intravenous injection of a synthetic analog of ACTH (250 μg). Subsequently, a single dose of metyrapone was administered at midnight and in the following morning, blood samples were collected for 2 h, followed by an intravenous injection of hydrocortisone (100 mg) with continued sampling. We show that increased cortisol serum levels secondary to ACTH stimulation or hydrocortisone administration are positively associated with plasma ghrelin levels, whereas central stimulation of the HPA axis by blocking cortisol synthesis with metyrapone is associated with decreased plasma ghrelin levels. Collectively, this suggests that HPA-axis-mediated elevations in ghrelin plasma concentration require increased peripheral cortisol levels, independent of central elevation of ACTH and possibly CRH levels.

Ghrelin is a growth hormone and cortisol secretagogue that plays an important role in appetite and weight regulation. It is not known whether ghrelin is involved in the eating response to stress in humans. In the present study we examined the effects of psychologically induced stress on plasma ghrelin levels in patients with binge-eating disorder (BED) (n = 8) and in healthy subjects of normal (n = 8) or increased (n = 8) body mass index (BMI). Volunteers were subjected to the standardized trier social stress test (TSST). Heart rate, blood pressure, serum cortisol, serum prolactin, and plasma ghrelin levels were measured throughout the test. In addition, subjects were requested to rate their feelings of anxiety, tension, urge to eat uncontrollably and desire to eat sweets by means of a visual analog scale both before and after the TSST There was a significant rise in the systolic blood pressure (p = 0.003) in the study population, reflecting induction of physiological changes by the psychological challenge. Basal ghrelin levels were higher in healthy normal weight (385.4 +/- 79pg/ml) than in obese (170.4 +/- 15.7pg/ml) subjects (p < 0.033). Basal ghrelin levels in patients with BED (240 +/- 40.8pg/ml) were at an intermediate level between thin and healthy obese subjects, but this difference did not attain statistical significance. There were no differences in ghrelin levels throughout the test among the groups after correction for BMI, age and gender. A significant difference in the trend time of ghrelin was revealed when the three groups were analyzed according to their cortisol response to stress. Ghrelin levels increased in cortisol responders whereas no change or a decrease in ghrelin levels occurred in cortisol non-responders (p = 0.038). Furthermore, a positive correlation was found between the change in ghrelin and the change in cortisol during TSST (r = 0.444, p = 0.029) but not between the change in ghrelin and the change in systolic blood pressure. The combined score of stress and anxiety was higher in subjects in the higher quartile of ghrelin response in comparison to the lower quartile both before (28.3 +/- 6.5 vs. 6.6 +/- 3.3, p=0.0077) and after (61.6 +/- 9 vs. 28.3 +/- 11.3, p = 0.033) TSST On the other hand, eating related scores did not differ according to quartiles of ghrelin response. Our findings indicate that a psychological stress may induce an increase in plasma ghrelin levels in humans, and that the post-stress induced urge for uncontrolled eating is not acutely modulated by stress related elevations in ghrelin levels. Furthermore, the stress induced increase in plasma ghrelin was associated with the acute response of serum cortisol to stress, but was independent of BMI or the presence of-BED. (c) 2007 Elsevier Ltd. All rights reserved.

BACKGROUND:Ghrelin is a potent GH secretagogue that also plays an important role in appetite and weight regulation. Ghrelin increases hunger and food intake, and its levels decrease after a standard meal or glucose. OBJECTIVE:To examine the effects of standard oral glucose, lipid and protein loads on ghrelin levels, investigating the possibility that these responses may be modulated by several anthropometric and metabolic factors. SUBJECTS AND METHODS:There were 24 adult nondiabetic subjects (13 men/11 women; mean age 55.3 +/- 2.9 years, range 26-74 years). Each participant underwent one or more of the following nutrient loads: (i) a standard oral glucose (75 g) load (n = 18); (ii) an oral lipid load (40 g, with 24 g saturated fat; n = 13); (iii) an oral protein load (40 g; n = 11). RESULTS:Fasting ghrelin levels were negatively related to body mass index (BMI; r =-0.47; P = 0.02), waist circumference (r = -0.58; P = 0.0028), waist/hip ratio (r = -0.56; P = 0.0046), fasting insulin (r = -0.44, P = 0.03), and homeostasis model assessment insulin resistance index (HOMA-R; r = -0.43, P = 0.034). Glucose load induced a decrease in ghrelin levels (P < 0.0001), and this response was modulated by sex (P < 0.0001), in that levels were significantly higher in females. The presence of obesity affected ghrelin response to glucose (< 0.0217), in that log-transformed ghrelin levels started to increase back to baseline after its initial decline earlier in obese than in lean subjects. Ghrelin levels after a glucose load were lower over time in subjects with more pronounced insulin resistance (P < 0.0001). Similarly, ghrelin levels decreased significantly following the lipid meal (P = 0.035), and were modulated by HOMA-R (P = 0.027) and gender (P = 0.029). Protein did not affect ghrelin levels. CONCLUSIONS:This study demonstrates that ghrelin levels respond in a different manner to glucose, lipid and protein loads, and are subject to modulation according to gender, obesity and insulin sensitivity.

The lipoxygenase pathway has been implicated in the growth, migration, and contraction of vascular smooth muscle cells (VSMCs). However, the precise type of lipoxygenase present in the vascular wall has not been characterized. In this study, we used a specific reverse-transcriptase polymerase chain reaction method with 2 sets of specific primers on total RNA and polyA (+)RNA of normal human VSMCs prepared from umbilical artery. Two forms of platelet-type 12-lipoxygenase mRNA were present in human VSMCs: the already published form cloned from human erythroleukemia cells and a variant form of platelet-type 12-lipoxygenase, which includes 2 additional sequences consistent with the 2 introns (D and E). This novel form of 12-lipoxygenase poly A (+)RNA was downregulated by lipopolysaccharide (10 &mgr;g/mL) and upregulated by epidermal growth factor (100 ng/mL) but was not affected by angiotensin II (10−7 mol/L). We developed a rabbit anti-human platelet-type 12-lipoxygenase polyclonal antibody directed against a 24-amino acid peptide encoded within exon 4. Western immunoblotting of protein extracted from VSMCs and umbilical artery and platelet extract with this antibody showed a coordinate 110-kDa protein and the already-described 70-kDa band detected in platelets and cord homogenate. Another 120-kDa protein was consistently detected in cord extracts but not in platelet or VSMC homogenates. The immunohistochemistry study performed with the same antibody showed extensive cytoplasmic staining of VSMCs. The specific role of these different forms of platelet-type 12-lipoxygenase is subject to further investigation.

The preparation of high quality plasmid DNA is a necessary requirement for most molecular biology applications. We compared four different large plasmid preparation protocols, which were based on either a liquid-phase approach (Triton lysis) or purification of alkaline lysis bacterial extracts followed by supercoiled plasmid purification on affinity columns. Two host Escherichia coli strains, JM 109 and INValphaF', were used to grow the test plasmids for comparison of product plasmid DNA produced from the four different plasmid isolation methods. While the DNA grown in E. coli strain JM109, prepared by liquid-phase Triton lysis was appropriately restricted by 12 restriction enzymes, this was not the case for any of the JM109-grown DNA purified by any of the affinity column solid-phase approaches. In contrast to this, when the plasmid DNA was grown in E. coli strain INValphaF', most restriction enzymes cut DNA appropriately, irregardless of the plasmid preparation protocol used. It seems that an impurity commonly eluted with the DNA from all three of the solid-phase DNA columns had an equal effect on the above enzymes using the common host strain JM109, but not strain INValphaF'.

In this study we examined the possibility that chronic reduction in serum dihydrotestosterone (DHT) attained by pharmacologic inhibition of 5 alpha-reductase modulates serum sex-hormone-binding globulin (SHBG) levels in elderly men. Twenty-one men, ages 58-79 years (mean 66) with benign prostatic hypertrophy were treated with the 5 alpha-reductase inhibitor finasteride (5 mg daily) for 12 months. Serum DHT declined by 80% (p < 0.001) and total testosterone rose by 14% (p < 0.05). Serum SHBG concentration remained unchanged (44.1 +/- 4.5 vs 45.2 +/- 5.7 nmol/l for pre- and post-therapy levels, respectively). Thus, the conversion of testosterone to DHT is not required to maintain the androgenic effect on serum SHBG concentration in elderly men.

OBJECTIVE Recent evidence suggests that androstanediol glucuronide (AG), a metabolite of dihydrotestosterone (DHT) formed in skin, is frequently elevated in hirsute women, presumably reflecting enhanced 5 alpha-reductase activity. An alternative method of demonstrating 5 alpha-reductase activity is the androsterone (A)/aetiocholanolone (E) ratio in urine. A and E are the 5 alpha- and 5 beta- reduced metabolites, respectively, of androstenedione, which is the principal metabolite of dehydroepiandrosterone (D). Although serum AG and the urinary A/E ratio have both been considered valid methods for assessing 5 alpha-reductase activity, the two have not been previously compared in hirsute women. The present study was undertaken to assess 5 alpha-reductase activity in hirsute patients as determined by these two different methods.PATIENTS AND MEASUREMENTS We surveyed 47 untreated women (ages 17-33) with various degrees of hirsutism. Serum testosterone, bioavailable testosterone, dehydroepiandrosterone sulphate, and AG were determined. Additionally, A, E and D were measured in 24-hour collections of urine.RESULTS For the 47 women, 37 had elevated blood levels of AG (17.4 +/- 2.2, mean +/- SEM; normal < 8 nmol/l), but only 18 of these had an increased urinary A/E ratio (> 1.5). All but one of the remainder had elevated urinary and/or serum androgen levels. Overall, no significant correlation between AG and A/E was observed. There was a highly significant correlation between AG in serum and A in urine (r=0.82, P < 0.001). AG was also positively related to dehydroepiandrosterone sulphate (r=0.64; P < 0.005), bioavailable testosterone (r=0.6; P<0.001), aetiocholanolone(r= 0.58; P < 0.001) and total testosterone (r= 0.52; P < 0.01). In contrast, A/E was not significantly related to androgen production. CONCLUSIONS There is a poor correlation between AG and the A/E ratio in hirsute women. Although AG may be raised by increased 5 alpha-reductase activity, it is probably also affected by the presence of elevated androgens regardless of 5 alpha-reductase activity.

Testosterone and androstenedione are metabolized by 5 alpha- and 5 beta-reductases to androsterone (A) and etiocholanolone (E), respectively. These are excreted in the urine as conjugates, and the A/E ratio in normal men is usually greater than or equal to 1.5 (as opposed to 1 in women) because of the high 5 alpha-reductase activity in the prostate. The A/E ratio can be determined simply by gas chromatography after acid hydrolysis of a urine sample, extraction of steroids, and formation of trimethylsilyl derivatives. A timed collection of urine is unnecessary because the ratio of A/E is used rather than absolute values. In men suffering from benign prostate hypertrophy who are treated with Finasteride (a 5 alpha-reductase inhibitor), the A/E ratio decreases to less than 0.5. The A/E ratio decrease can be detected long before there is clinical improvement.

Solid-phase chemiluminescence based immunoassay methods for the measurement of steroids and urinary steroid conjugates are described. The assays incorporate the following features: (i) a steroid-chemiluminescent marker conjugate serves as the labeled ligand; (ii) polyclonal or monoclonal antibodies covalently coupled to polymer beads are utilized as the specific immunoadsorbent; and (iii) a centrifugation step after the binding reaction is used to remove interferring luminescent compounds along with unbound steroid. These assays proved satisfactory with respect to sensitivity, precision and accuracy and offer significant advantages over existing methods in speed, safety and economy. Moreover, the use of monoclonal antibodies provides optimal specifity and rigorous standardization.

Solid-phase chemiluminescence based immunoassay methods for the measurement of steroids and urinary steroid conjugates are described. The assays incorporate the following features: (i) a steroidchemiluminescent marker conjugate serves as the labeled ligand; (ii) polyclonal or monoclonal antibodies covalently coupled to polymer beads are utilized as the specific immunoadsorbent; and (iii) a centrifugation step after the binding reaction is used to remove interferring luminescent compounds along with unbound steroid. These assays proved satisfactory with respect to sensitivity, precision and accuracy and offer significant advantages over existing methods in speed, safety and economy. Moreover, the use of monoclonal antibodies provides optimal specifity and rigorous standardization.