Marco Mezzullo

Fecal metabolome of the Hadza hunter-gatherers: A host-microbiome integrative view

The recent characterization of the gut microbiome of traditional rural and foraging societies allowed us to appreciate the essential co-adaptive role of the microbiome in complementing our physiology, opening up significant questions on how the microbiota changes that have occurred in industrialized urban populations may have altered the microbiota-host co-metabolic network, contributing to the growing list of Western diseases. Here, we applied a targeted metabolomics approach to profile the fecal metabolome of the Hadza of Tanzania, one of the world’s few remaining foraging populations, and compared them to the profiles of urban living Italians, as representative of people in the post-industrialized West. Data analysis shows that during the rainy season, when the diet is primarily plant-based, Hadza are characterized by a distinctive enrichment in hexoses, glycerophospholipids, sphingolipids, and acylcarnitines, while deplete in the most common natural amino acids and derivatives. Complementary to the documented unique metagenomic features of their gut microbiome, our findings on the Hadza metabolome lend support to the notion of an alternate microbiome configuration befitting of a nomadic forager lifestyle, which helps maintain metabolic homeostasis through an overall scarcity of inflammatory factors, which are instead highly represented in the Italian metabolome.

Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro

BackgroundHuman luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG.MethodsCultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated.ResultsWe found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis.ConclusionsThese data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.

Comparison of three patterns of feed supplementation with live Saccharomyces cerevisiae yeast on postweaning diarrhea, health status, and blood metabolic profile of susceptible weaning pigs orally challenged with Escherichia coli F4ac.

The development of effective feeding strategies to reduce the detrimental effect of enterotoxigenic F4ac (ETEC) plays a crucial role in reducing the occurrence of therapeutic intervention with antibiotics in livestock. The ability of CNCM I-4407 (SCC), supplied in different patterns to counteract ETEC infection in weaned pigs, was evaluated. Fifty pigs weaned at 24 d were then divided into 5 groups: control (CO), CO + colistin (AB), CO + 5 × 10(10) cfu of SCC/ kg feed, from d 0 to 21 (PR), CO + 5 × 10(10) cfu of SCC/ kg feed from d 7 to 11 (CM), and CO + 1 shot of 2 × 10(11) cfu of SCC when the first diarrhea appeared (CU). On d 7 postweaning, all the pigs were orally challenged with 10(8) cfu of ETEC. Blood samples were taken from the pigs (d 7, 8, 12, and 21) while the fecal excretion of ETEC was assessed on d 7 and 10. Fecal consistency was scored from 12 h before infection to 144 h postinfection (p.i.). On d 21, the pigs were sacrificed. The in vitro adhesion test on the intestinal villi confirmed individual susceptibility to ETEC, excluding the presence of resistant pigs. Growth performance did not differ between the treatments. Mortality was reduced in the AB group (P< 0.01) and, marginally, in the PR group (P = 0.089) when compared to the CO group. The CO group had a higher fecal score than AB in the period of observation (from P = 0.01 to P< 0.001). Yeast administration reduced the fecal score when compared to the CO group 12 and 48 h p.i. (P = 0.04). Total IgA never differed among the treatments, but the ETEC-specific IgA concentration was lower in the AB group than in CO (P = 0.04) at d 12. Four days p.i., the pigs fed live yeast had reduced ETEC excretion compared with the CO pigs (P = 0.05). Blood concentrations of dodecenoyl-L-carnitine (P < 0.01), glutaryl-L-carnitine/hydroxyhex¬anoyl-L-carnitine, phosphatidylcholine diacyl and phosphatidylcholine diacyl (P = 0.01 and P< 0.01, respectively), and α-amino adipic acid (P < 0.01) were reduced in the AB group compared to the CO group; PR + CM reduced the concentration of sphingomyelin-ceramide (P = 0.02) and increased the concentration of decadienyl-L-carnitine (C10:2; P= 0.02) vs. CO. The CM group had an increased concentration of C10:2 (P < 0.01) compared to the PR group. In conclusion, the administration of live yeast, even in concomitance with ETEC infections, reduces pig illness and mortality. The strain of SCC tested did not show a therapeutic effect.

Validation of an LC–MS/MS salivary assay for glucocorticoid status assessment: Evaluation of the diurnal fluctuation of cortisol and cortisone and of their association within and between serum and saliva

Salivary steroid testing represents a valuable source of biological information; however, the proper measurement of low salivary levels is challenging for direct immunoassays, lacking adequate sensitivity and specificity and causing poor inter-laboratory reproducibility. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has overcome previous analytical limits, often providing results deviating from previous knowledge. Nowadays, LC-MS/MS is being introduced in clinical laboratories for salivary cortisol testing; however, so far only a few studies have reported thorough biological validation based on LC-MS/MS data. In this study, we provide a thorough analytical, pre-analytical and biological validation of an LC-MS/MS method for the measurement of salivary cortisol (F) and of its inactive metabolite cortisone (E). Analytes were extracted from 50μl of saliva, were then separated in 7.5min LC-gradient and detected by negative electrospray ionization-multiple reaction monitoring. The reliability of a widely diffused collection device, Salivette(®), was assessed and the overall procedure was validated. The diurnal cortisol and cortisone fluctuation in saliva and serum was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. The assay allowed the quantitation of F and E down to 39.1 and 78.1pg/ml, with an imprecision range of 5.5-9.5%, 3.9-14.1% and 2.6-14.4%, and an accuracy range of 105.5-113.1%, 88.5-98.7% and 90.7-96.7% for both analytes at low, medium and high levels, respectively. Salivette(®) provided comparable results and better precision (CV<1.0%) as referred to direct spitting (CV<13.0%). A parallel diurnal rhythm in saliva and serum was observed for cortisol and cortisone, with values lowering from the morning to the evening time points (P<0.0001). While salivary E linearly correlated to total serum F (R(2)=0.854, P<0.001), salivary F showed an exponential relationship (R(2)=0.903, P<0.001) with serum F reflecting the free circulating fraction. A non linear association between E and F was observed in saliva (R(2)=0.941, p<0.001) consistent with the type II 11β-HSD activity. We concluded that our LC-MS/MS method allowed a sensitive evaluation of salivary levels of cortisol and cortisone. The simultaneous determination of both hormones in saliva allowed the differential estimation of the active and of the total glucocorticoid exposure over the daytime. The assay could provide further insight into the comprehension of normal and dysfunctional glucocorticoid circadian rhythm.

Acute severe male hypo-testosteronemia affects central motor command in humans

PURPOSE To indirectly evaluate the effect of androgens on neuromuscular system in humans we analyzed if an induced short-term hypogonadal state (serum total testosterone-TT<2.3ng/ml) may affect central drive to skeletal muscle and/or muscle neuro-mechanical performance. METHODS We compared voluntary and electrically evoked muscle sEMG signals from biceps brachii in nine hypogonadal male volunteers (Hypo) and in ten healthy controls (Cont). Serum TT and dihydrotestosterone (DHT) were assayed. RESULTS With respect to Hypo, Cont exhibited significantly higher median frequency content (MDF) at any angular velocity; normalized MDF [95.9% (SD=23.3) vs 73.8% (SD=9.3)]; muscle fiber conduction velocity (CV) from lowest to highest angular velocities; initial MDF at fatigue test [91.78Hz (SD=22.03) vs 70.94Hz (SD=11.06)] as well as was the normalized slope [-0.64 (SD=0.14 vs -0.5 (SD=0.11)]. In the non-fatigued state, Hypo showed a slower single twitches time to peak (TTP). In Cont, half relaxation time (HRT) decreased after fatigue while increased in Hypo (p<0.05 between groups). A significant correlation between both TT and dihydrotestosterone with MDF and CV was found during voluntary contractions only. CONCLUSIONS A brief exposure to very low serum TT concentration in males seem to determine a reduced excitability of the NM system which, in turn, would favor a predominant recruitment of slow twitch MUs.

Human chorionic gonadotropin stimulation gives evidence of differences in testicular steroidogenesis in Klinefelter syndrome, as assessed by liquid chromatography-tandem mass spectrometry.

BACKGROUND Men with Klinefelter syndrome (KS) show hypergonadotropic hypogonadism, but the pathogenesis of hypotestosteronemia remains unclear. Testicular steroidogenesis in KS men was evaluated over three decades ago after human chorionic gonadotropin (hCG) stimulation, but inconclusive results were obtained. Intriguingly, some recent studies show increased intratesticular testosterone concentrations in men with KS. OBJECTIVE To analyze serum steroid profile, as a proxy of testicular steroidogenesis, after hCG stimulation in KS compared with control men. DESIGN A prospective, longitudinal, case-control, clinical trial. METHODS Thirteen KS patients (36±9 years) not receiving testosterone (TS) replacement therapy and 12 eugonadic controls (32±8 years) were enrolled. Serum steroids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at baseline and for five consecutive days after intramuscular injection of 5000IU hCG. RESULTS Progesterone (P), 17-hydroxyprogesterone (17OHP), TS, and estradiol (E2) showed a significant increase (P<0.001) after hCG stimulation in both groups. On the contrary, androstenedione (AS) and dehydroepiandrosterone did not increase after hCG stimulation. The 17OHP/P ratio increased in both groups (P<0.001), the TS/AS ratio (17β-hydroxysteroid dehydrogenase type 3 (17βHSD3) activity) did not increase after hCG in any group, and the E2/TS ratio (aromatase activity) increased significantly in both groups (P=0.009 in KS and P<0.001 in controls). Luteinizing hormone decreased after hCG in both groups (P=0.014 in KS and P<0.001 in controls), whereas follicle-stimulating hormone decreased only in control men (P<0.001). CONCLUSION This study demonstrates for the first time using LC-MS/MS that Leydig cells of KS men are able to respond to hCG stimulation and that the first steps of steroidogenesis are fully functional. However, the TS production in KS men is impaired, possibly related to reduced hydroxysteroid deydrogenase activity due to an unfavorable intratesticular metabolic state.

Clomiphene citrate effect in obese men with low serum testosterone treated with metformin due to dysmetabolic disorders: A randomized, double-blind, placebo-controlled study

Context Low testosterone (T) levels are often found in obese men with impaired glucose tolerance (IGT) and overt type 2 diabetes (T2DM); however, the mechanisms underlying this condition and its correct therapy are still under debate. Objective To evaluate the effectiveness of clomiphene citrate (CC) in increasing endogenous T levels in obese men with low serum T and with IGT or T2DM treated with metformin (MET). Design Cross-over, randomized, double-blind, placebo-controlled study. Methods 24 obese men, aged 47.3 ±. 6.3 (range 35–55 years), with low T level (≤3 ng/mL) and naïve diagnosis of IGT or T2DM were included. Subjects were randomized to CC 25 mg/day or placebo (Plac) with MET 2 g/day for 3 months. After a 6-week wash-out period, subjects were moved to the alternative arm for additional 3 months. Clinical evaluation and blood exams performed prior to and at the end of treatment. Results Of 24 randomized, 21 were evaluable, classified as IGT (n = 11) or T2DM (n = 10). Compared to baseline levels, T levels increased significantly after 3 months of CC treatment (3.03±0.80 to 5.99±1.67 ng/mL P<0.001) but not after the Plac treatment (2.87±0.78 to 3.09±0.84 ng/mL P<0.001 between the treatments). T changes were similar in IGT and T2DM subjects. Gonadotropins as well raised significantly after CC treatment (LH 3.83±1.45 to 8.53±6.40 mU/mL; FSH 4.84±1.67 to 10.15±5.08 mU/mL P<0.001 respectively), whereas no changes for LH (3.51±1.59 to 3.63±1.39 mU/mL) but a smooth increased for FSH (4.61±2.49 to 5.39±2.65 mU/mL; P = 0.004) were shown after Plac treatment (LH P = 0.001 and FSH P = 0.002 between treatments). Furthermore, fasting glucose (106.8±23.2 to 101.1±25.7 mg/dL; P = 0.004), insulin (19.3±12.1 to 15.6±10.1 μU/mL; P = 0.010) and HOMA-IR (4.94±2.89 to 3.69±2.12; P = 0.001) decreased significantly during the CC treatment period, whereas no significant changes were observed in any of these parameters in the Plac treatment. Conclusions A low dose of CC therapy was able to significantly increase serum T levels in all participants with mild modifications of clinical and metabolic parameters. Trial registration EudraCT 2011-000439-10

Parallel diurnal fluctuation of testosterone, androstenedione, dehydroepiandrosterone and 17OHprogesterone as assessed in serum and saliva: validation of a novel liquid chromatography-tandem mass spectrometry method for salivary steroid profiling

Abstract Background: Salivary androgen testing represents a valuable source of biological information. However, the proper measurement of such low levels is challenging for direct immunoassays, lacking adequate accuracy. In the last few years, many conflicting findings reporting low correlation with the serum counterparts have hampered the clinical application of salivary androgen testing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it possible to overcome previous analytical limits, providing new insights in endocrinology practice. Methods: Salivary testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA) and 17OHprogesterone (17OHP) were extracted from 500µL of saliva, separated in 9.5 min LC-gradient and detected by positive electrospray ionization – multiple reaction monitoring. The diurnal variation of salivary and serum androgens was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. Results: The assay allowed the quantitation of T, A, DHEA and 17OHP down to 3.40, 6.81, 271.0 and 23.7 pmol/L, respectively, with accuracy between 83.0 and 106.1% for all analytes. A parallel diurnal rhythm in saliva and serum was observed for all androgens, with values decreasing from the morning to the evening time points. Salivary androgen levels revealed a high linear correlation with serum counterparts in both sexes (T: R>0.85; A: R>0.90; DHEA: R>0.73 and 17OHP: R>0.89; p<0.0001 for all). Conclusions: Our LC-MS/MS method allowed a sensitive evaluation of androgen salivary levels and represents an optimal technique to explore the relevance of a comprehensive androgen profile as measured in saliva for the study of androgen secretion modulation and activity in physiologic and pathologic states.

Metabolomics evidences plasma and serum biomarkers differentiating two heavy pig breeds.

In pigs, many production traits are known to vary among breeds or lines. These traits can be considered end phenotypes or external traits as they are the final results of complex biological interactions and processes whose fine biological mechanisms are still largely unknown. This study was designed to compare plasma and serum metabolomic profiles between animals of two heavy pig breeds (12 Italian Large White and 12 Italian Duroc), testing indirectly the hypothesis that different genetic backgrounds might be the determining factors of differences observed on the level of metabolites in the analyzed biofluids between breeds. We used a targeted metabolomic approach based on mass spectrometric detection of about 180 metabolites and applied a statistical validation pipeline to identify differences in the metabolomic profiles of the two heavy pig breeds. Blood samples were collected after jugulation at the slaughterhouse and prepared for metabolomics analysis that was carried out using the Biocrates AbsoluteIDQ p180 Kit, covering five different biochemical classes: glycerophospholipids, amino acids, biogenic amines, hexoses and acylcarnitines. A statistical pipeline that included the selection of the most relevant metabolites differentiating the two breeds by sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was coupled with a stability test and significance test determined with leave one out and permutation procedures. sPLS-DA plots clearly separated the pigs of the two investigated breeds. A few metabolites (a total of five metabolites considering the two biofluids) involved in key metabolic pathways largely contributed to these differences between breeds. In particular, a higher level of the sphingomyelins SM (OH) C14:1 (both in plasma and serum), SM (OH) C16:1 (in serum) and SM C16:0 (in serum) were observed in Italian Duroc than in Italian Large White pigs and the inverse was for the biogenic amine kynurenine (in plasma). The level of another biogenic amine (acetylornithine) was higher in Italian Large White than in Italian Duroc pigs in both analysed biofluids. These results provided biomarkers that could be important to understand the biological differences between these two heavy pig breeds. In particular, according to the functional role played by sphingomyelins in obesity-induced inflammatory responses, it could be possible to speculate that a higher level of sphingomyelins in Italian Duroc might be related to the higher interrmuscular fat deposition of this breed compared with the Italian Large White. Additional studies will be needed to evaluate the relevance of these biomarkers for practical applications in pig breeding and nutrition.

Central hypogonadism due to a giant, “silent” FSH-secreting, atypical pituitary adenoma: effects of adenoma dissection and short-term Leydig cell stimulation by luteinizing hormone (LH) and human chorionic gonadotropin (hCG)

Abstract We present a case report of an atypical giant pituitary adenoma secreting follicle-stimulating hormone (FSH). A 55-year-old patient presented for erectile dysfunction, loss of libido and fatigue. The biochemical evaluation showed very high FSH serum levels in the presence of central hypogonadism. Neither testicular enlargement nor increased sperm count was observed, thus a secretion of FSH with reduced biological activity was supposed. The histological examination after neuro-surgery showed an atypical pituitary adenoma with FSH-positive cells. Hypogonadism persisted and semen analyses impaired until azoospermia in conjunction with the reduction in FSH levels suggesting that, at least in part, this gonadotropin should be biologically active. Thus, we hypothesized a concomitant primary testicular insufficiency. The patient underwent short-term treatment trials with low doses of either recombinant luteinizing hormone (LH) or human chorionic gonadotropin (hCG) in three consecutive treatment schemes, showing an equal efficacy in stimulating testosterone (T) increase. This is the first case of atypical, giant FSH-secreting pituitary adenoma with high FSH serum levels without signs of testicular hyperstimulation, in presence of hypogonadism with plausible combined primary and secondary etiology. Hypophysectomized patients may represent a good model to assess both pharmacodynamics and effective dose of LH and hCG in the male.