SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. This report is the summary of the results of that trial. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n=67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene® Blood RNA tubes; n=35). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24h after the first extraction for the second tube. Participants (n=93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1ß, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a critical performance of the laboratory, 33% of the laboratories were included in this group. The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of RNA analysis of blood samples.

How standardization of the pre-analytical phase of both research and diagnostic biomaterials can increase reproducibility of biomedical research and diagnostics.

Comparison of published biomedical studies shows that a large proportion are irreproducible, causing severe damage to society and creating an image of wasted investments. These observations are of course damaging to the biomedical research field, which is currently full of future promise. Precision medicine and disease prevention are successful, but are progressing slowly due to irreproducible study results. Although standardization is mentioned as a possible solution, it is not always clear how this could decrease or prevent irreproducible results in biomedical studies. In this article more insight is given into what quality, norms, standardization, certification, accreditation and optimized infrastructure can accomplish to reveal causes of irreproducibility and increase reproducibility when collecting biomaterials. CEN and ISO standards for the sample pre-analytical phase are currently being developed with the support of the SPIDIA4P project, and their role in increasing reproducibility in both biomedical research and diagnostics is demonstrated. In particular, it is described how standardized methods and quality assurance documentation can be exploited as tools for: 1) recognition and rejection of 'not fit for purpose' samples on the basis of detailed sample metadata, and 2) identification of methods that contribute to irreproducibility which can be adapted or replaced.

Longevity index (LI%) and centenarity index (CI%): new indicators to evaluate the characteristics of aging process in the Italian population.

Centenarians represent a group of population displaying the most rapid expansion. This progressive increase of the presence of centenarians is a multi-factorial phenomenon, which is due to the improvements of the environmental conditions and the life style and also to the progress of the medical science. In order to obtain a more reliable estimate of the longevity per gender and territorial entities, we propose two new indicators. (i) The ratio between the ultranonagenarians and the total population above 65 years old (called longevity index: LI%); (ii) the ratio between the centenarians and the total population above 90 years old (called centenarity index: CI%). An analysis of the data of the Italian National Office of Statistics (ISTAT, ) using these two indicators demonstrates that the subjects above the age of 90 are more frequent in the regions of Central and Northern Italy, which are more developed regarding the economic conditions and technological progress. Nevertheless, the Southern and Insular regions of Italy have a higher occurrence of centenarians among the ultranonagenarian population, and also a higher prevalence of male centenarians, as compared to the northern regions. This demonstrates that achievement of the threshold of 100 years old does not require only particular socio-economic conditions, but also an adequate climate and environment, as well as a favorable genetic composition.

Laser-assisted microdissection for real-time PCR sample preparation.

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.

Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat.

It has been demonstrated that the forebrain cholinergic system and the extracellular regulated kinase signal transduction pathway are involved in the mechanisms of learning, encoding, and storage of information. We investigated the involvement of the cholinergic and glutamatergic systems projecting to the medial prefrontal cortex and ventral hippocampus and of the extracellular regulated kinase signal transduction pathway in the acquisition and recall of the step-down inhibitory avoidance response in the rat, a relatively simple behavioral test acquired in a one-trial session. To this aim we studied by microdialysis the release of acetylcholine and glutamate, and by immunohistochemistry the activation of extracellular regulated kinase during acquisition, encoding and recall of the behavior. Cholinergic, but not glutamatergic, neurons projecting to the medial prefrontal cortex and ventral hippocampus were activated during acquisition of the task, as shown by increase in cortical and hippocampal acetylcholine release. Released acetylcholine in turn activated extracellular regulated kinase in neurons located in the target structures, since the muscarinic receptor antagonist scopolamine blocked extracellular regulated kinase activation. Both increased acetylcholine release and extracellular regulated kinase activation were necessary for memory formation, as administration of scopolamine and of extracellular regulated kinase inhibitors was followed by blockade of extracellular regulated kinase activation and amnesia. Our data indicate that a critical function of the learning-associated increase in acetylcholine release is to promote the activation of the extracellular regulated kinase signal transduction pathway and help understanding the role of these systems in the encoding of an inhibitory avoidance memory.

Comparison of telomerase activity in bladder carcinoma and exfoliated cells collected in urine and bladder washings, using a quantitative assay.

Telomerase activity was measured with a quantitative assay, based on a modification of telomeric repeat amplification protocol method, in bladder cancers and apparently normal mucosa in 33 patients. In the same patients, the enzyme was also measured in exfoliated cells collected both with voided urine and bladder washings. Results obtained in urine were compared with those from 20 healthy subjects. Telomerase activity was present in 31 (94%) bladder cancer tissues and in 23 (72%) apparently normal mucosa samples. However, the levels of enzyme activity were significantly higher in cancer tissues in comparison with normal mucosa (mean +/- SD, 47.3 +/- 23.2 and 14.9 +/- 6.1 ng DNA/microg protein, respectively; P < 0.0001). Telomerase activity in bladder cancer tissues was not related to tumor stage and grade. Enzyme activity was present in 27 urine samples and in 27 (82%) bladder washings collected from cancer patients. We did not find correlation between the activity in urine and washings, and their mean levels were not different (22.2 +/- 10.1 and 20.7 +/- 8.0, respectively). Telomerase activity in bladder cancer tissues was correlated to its activity in urine (r = 0.650, P < 0.001) and in bladder washings (r = 0.410, P < 0.05). Only 2 of 20 urine samples from control subjects were found to express telomerase activity at a very low level. This was the first attempt to correlate telomerase activity in exfoliated cells from urine and bladder washings with the activity in corresponding bladder cancers. According to these results we postulate that telomerase activity in urine sediment reflects the activity in bladder cancers better than bladder washings and, for its easy collection, is to be preferred as diagnostic marker in this tumor.

Quantitation of somatostatin receptor type 2 gene expression in neuroblastoma cell lines and primary tumors using competitive reverse transcription-polymerase chain reaction.

We previously reported the presence of somatostatin (SS-14)-binding sites in a wide panel of human neuroblastoma (NB) tumor cell lines. Given that the adrenal gland and its relative embryonal and adult tumors express an abundance of mRNA for somatostatin receptor type 2 (sst2) mRNA, we studied the quantitative expression of sst2 in 6 NB cell lines and 15 primary tumors using competitive reverse transcription (RT)-PCR. This method uses an insertion mutant of the target gene as a competitor for the RT-PCR reaction, thus allowing exact quantitation of sst2 mRNA abundance. We found expression of specific transcripts for sst2 in all of the NB cell lines and tumors investigated (range, 9 x 10(5)-4 x 10(9) molecules/microg RNA). In NB cells, the expression of sst2 was highly correlated with SS-14-binding sites (R = 0.93). In primary tumors, sst2 was positively related to the expression of the neuroendocrine marker secretogranin II (P < 0.05) and negatively related to N-myc amplification (a poor prognostic factor, P < 0.005) and metastatic dissemination (P < 0.05). In addition, Kaplan-Meier curves indicate that sst2 expression is positively related to survival (P = 0.01). In a patient with stage IVs disease (a spontaneously regressing form), we found the highest sst2 expression (4 x 10(9) molecules/microgram RNA), a value relatively similar to that of normal adrenal. In conclusion, these data indicate that quantitation of sst2, as assessed with competitive RT-PCR, could represent a new prognostic tool in the neuroendocrine tumor NB. Since sst2 recognizes octreotide with high affinity, these findings could also have both diagnostic and therapeutic value.

Effect of diet and red wine consumption on serum total antioxidant capacity (TAC), dehydroepiandrosterone-sulphate (DHEAS) and insulin-like growth factor-1 (IGF-1) in Italian centenarians.

The traditional mediterranean diet is associated with a hope for longer survival. It has also been shown that the red wine possesses a protective effect against the oxidative stress. We studied TAC, the DHEAS and the IGF-1 in a group of 26 healthy centenarians, 17 women and 9 men, of the age range of 100--105 years. Furthermore, we analyzed also serum urate and bilirubin levels between drinkers and abstainers. Most of centenarian subjects have been moderate wine consumers (<500 ml/day of red wine). These subjects were subdivided as follows: (i) Group A: those who had maintained the style of their dietary habits as compared to the previous years (n=3 males, 10 females); (ii) Group B: those who actually consumed a diet being deficient compared to that of the previous years, but remained moderate drinkers of red wine (n=3 males, 4 females); and (iii) Group C: those who actually consumed a diet being deficient compared to that of the previous years, and at the same time, were abstainers in wine consumption (n=3 males, 3 females). The results show that in men three of the studied parameters decreased from Group A to C to considerable extents, as follows (mean+/-S.D.). TAC: 302.4+/-32.3; 142.0+/-24.1 and 96.4+/-20.1 micromol/l; DHEAS: 3.35+/-0.81; 2.52+/-0.18 and 1.34+/-0.14 micromol/l; IGF-1: 85.7+/-6.7; 76.6+/-6.7 and 65.6+/-2.6 ng/ml, respectively. For the same parameters, the results in the women were: TAC: 258.4+/-12.2; 182.1+/-14.0 and 107.6+/-10.0 micromol/l; DHEAS: 3.85+/-0.16; 2.34+/-0.19 and 2.05+/-0.04 micromol/l; IGF-1: 89.7+/-6.7; 76.6+/-4.7 and 64.2+/-2.7 ng/ml, respectively. We did not find any significant difference in the other serum parameters between drinkers (n=14) and abstainers (n=3) (urate: 267.6+/-52.9, and 289.5+/-80.1; bilirubin: 9.81+/-4.29 and 7.18+/-2.89 micromol/l, respectively). Our data suggest that the deteriorated diet caused a reduction of TAC, DHEAS and IGF-1 in the centenarians. However, red vine consumption exerted a protective effect against this trend, even if this protection is not reaching statistical significance in some cases (in men), which is due most probably to the lower number of male subjects in the study.

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience.

BACKGROUND: DNA integrity is a critical part of the definition of genomic DNA (gDNA) quality and can influence downstream molecular applications. Pre-analytical variables as sample storage and DNA extraction methods can influence the quality and quantity of isolated DNA and affect molecular test performances. The aim of this paper is to investigate the role of blood sample storage and DNA extraction procedures on gDNA integrity and gDNA fragmentation impact on a molecular test. METHODS: 157 DNA samples deriving from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. 157 DNA samples derived from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), which aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. RESULTS/CONCLUSIONS: Our results demonstrate that blood sample storage and DNA extraction procedures influence gDNA integrity and that the same blood sample which underwent a long range multiplex PCR based analytical test can provide different results if the adopted pre-analytical procedures are not standardized.

Measurement of somatostatin receptor subtype 2 mRNA in breast cancer and corresponding normal tissue.

Somatostatin analogs are effective in inhibiting growth of human breast cancer cell lines. These antiproliferative effects are mediated by specific receptors located on cell membranes. The somatostatin receptor subtype 2 (sst2) is the principal mediator of somatostatin effects in normal and cancer cells, and its presence has already been demonstrated in breast cancer. The purpose of our study was to evaluate the clinical relevance of the expression of sst2 by quantifying its mRNA in a large group of infiltrating breast cancers and their corresponding normal tissues. The expression of sst2 mRNA was measured with quantitative real time RT-PCR in 169 breast cancers and in their corresponding unaffected tissues. We evaluated the association of sst2 expression with the commonest clinical-pathologic features of breast cancer. The correlation with a marker of cell proliferation (Ki-67) and with receptor concentration was also evaluated. In cancer tissues, we found that the absolute concentrations of sst2 mRNA were significantly higher in estrogen receptor (ER)-positive samples (P=0.002) as well as in lymph-node-negative cancers (P=0.04) (Student's t-test or one-way ANOVA). In addition, sst2 mRNA was significantly higher in breast cancers than in corresponding unaffected tissues (P=0.0002). However, when the clinical-pathologic parameters were considered, this gradient maintained its statistical significance only in tumors expressing positive prognostic markers, such as the presence of ER (P=0.0005) and progesterone receptors (PgR) (P=0005), and the lack of lymph-node involvement (P=0.0003). The same difference was also significant in postmenopausal women (P=0.001) and in T1 patients (P=0.001). In addition, sst2 mRNA expression was significantly higher (P=0.008) in low-proliferating breast cancers. Finally, we found that the quantitative expression of sst2 mRNA was directly related to the PgR concentration in breast cancer tissues (P<0.001). Our data seem to indicate that an upregulation of sst2 gene expression is a common feature of breast cancers which, on the basis of conventional predictive parameters, are expected to have a better prognosis. Featuring a possible role of somatostatin analogs in combined endocrine therapies for breast cancer, our results seem to confirm that the sst2 status of the tumor should be previously investigated.

Telomerase activity is significantly enhanced in malignant adrenocortical tumors in comparison to benign adrenocortical adenomas.

Telomerase is an enzyme that causes short repeated sequence addition to the ends of chromosomes, thereby preventing their shortening during cell division and counteracting cell senescence. Telomerase activity is generally absent in adult differentiated cells, whereas it has been demonstrated in tumor cells, suggesting that its presence might be considered an index of malignancy. To evaluate whether telomerase might be considered a good predictive index of malignancy in adrenocortical tumors, we measured telomerase activity in 11 adrenal adenomas and 7 carcinomas obtained at surgery, using an original quantitative method. Telomerase activity was significantly higher (P<0.001) in carcinomas than in adenomas (median, 15.2 ng DNA/microg protein; range, 9.0-27.6 vs. 2.0; range, 0-8.3), and no overlap was observed between the 2 groups. In carcinomas, telomerase activity was significantly correlated with tumor diameter (r = 0.939; P<0.0001), whereas in adenomas it was not. The results of this study suggest that quantitative telomerase measurement may represent a useful tool to differentiate malignant from benign adrenocortical tumors.

Quantitative determination of sst2 gene expression in neuroblastoma tumor predicts patient outcome.

Neuroblastoma (NB) is the most common pediatric neuroendocrine tumor, and it is characterized by a quite variable clinical course. We previously found a great variability in the expression of somatostatin receptor type 2 (sst2) in several human NB cell lines and primary tumors. In this report we investigated whether expression of sst2 is somehow related to clinical outcome. We performed a retrospective study on 54 patients with a maximum follow-up of 100 months. The concentration of specific messenger ribonucleic acid (mRNA) for sst2 was measured by competitive RT-PCR and validated, in a small subset of samples, by quantitative imaging of gene (in situ hybridization) and protein (immunohistochemistry) expression. We found that sst2 mRNA was variably expressed in all NB tumors (range, 2.5 x 10(5) to 8 x 10(9) molecules/microg RNA) with a relevant reduction in the more advanced stage (P < 0.01). Analysis of Kaplan-Meier curves indicated that sst2 expression is positively related to the overall (P < 0.0001) and event-free (P < 0.0001) survival. Expression of sst2 was negatively related to tumor stage (P < 0.02) and MYCN amplification (P < 0.001), a poor prognostic factor. However, the prognostic information derived from sst2 is apparently independent from MYCN amplification, as assessed by stratifying sst2 values according to MYCN. In addition, the expression of sst2 was the only significant prognostic factor (P < 0.02) when it was included in a multivariate model containing other well known prognostic factors such as age, stage, and MYCN amplification. Hence, we propose that sst2 expression represents a new prognostic marker for NB. The main clinical value of a quantitative measure of sst2 lies in its ability to detect patients at low risk, independently from other prognostic factor, including MYCN amplification.

In vivo amino acid release from the striatum of aging rats: adenosine modulation.

The release of glutamate, aspartate, GABA, and taurine from the striatum of young (3 months), mature (12 months), and old (22 months), freely moving male rats was investigated by using a microdialysis fiber inserted transversally in the striatum. In old rats basal extracellular glutamate and aspartate levels were decreased vs. young rats (-38 and -49%, respectively). GABA and taurine levels were unmodified by age. In the presence of the adenosine receptor antagonist 8-phenyltheophilline (8-pT) at the concentration of 50 microM, both K(+)-evoked releases of glutamate and aspartate were more than doubled in young, but not in mature and old rats. 8-pT at the concentration of 500 microM significantly decreased glutamate basal levels and K(+)-evoked aspartate release in old rats only. GABA and taurine releases were not affected by 8-pT at either dose. Our findings indicate a modified adenosine modulation on glutamate and aspartate release in aged rats, that could result from a change in the balance between A1 and A2a adenosine receptor density or an alteration of A1 and A2a receptor-effector coupling.

Immunometric assay for lutropin (hLH) based on the use of universal reagents for enzymatic labelling and magnetic separation and monitored by enhanced chemiluminescence.

A solid-phase immunometric assay of human lutropin (hLH) is described. Two different anti-hLH antibodies were utilized as capture antibodies, and anti-IgG antibodies covalently coupled to magnetic particles and horseradish peroxidase, respectively, served as 'universal' detection reagents. An anti-hLH antibody raised in rabbits was incubated with a goat anti-rabbit IgG covalently bound to magnetic particles. The resulting complex was added to a separately incubated mixture of hLH and monoclonal anti-hLH antibody. Following incubation, the immunocomplex was sedimented in a magnetic field and the supernatant discarded. Finally a sheep anti-mouse antibody (F(ab')2 fragment) conjugated to horseradish peroxidase as label was added. Following a further incubation, the particles were sedimented in the magnetic field and washed. The hLH content of the sample was quantitated by measuring 'enhanced chemiluminescence'. The sensitivity of the assay was 2.5 +/- 0.9 IU/l (mean +/- SD), the within-run variation ranged from 7.9 to 11%, the between-run variation from 12.9 to 19.8%. Cross-reaction with hFSH or hTSH could not be detected, but was approximately 0.1% with hCG. The results correlated well with those obtained by radioimmunoassay (r = 0.84).

Cholinergic and noradrenergic denervations decrease labelled purine release from electrically stimulated rat cortical slices.

The origin of cortical purine release was investigated by measuring [3H]purine release from electrically stimulated cortical slices of rats after neurotoxic lesions of cholinergic, noradrenergic and serotoninergic pathways innervating the cortex. Purines were labelled by incubating the cortical slices with [3H]adenine. The 3H efflux at rest and during stimulation, analysed by high performance liquid chromatography, consisted of adenosine, inosine, hypoxanthine and a small amount of nucleotides. Twenty days after unilateral or bilateral lesion of the nucleus basalis a marked decrease in choline acetyltransferase activity was associated with a decrease in [3H]purine release. A linear relationship was found between the decrease in choline acetyltransferase activity and [3H]purine release. A partial recovery in both choline acetyltransferase activity and [3H]purine release was observed eight months after the lesion. Twenty days after intra-cerebroventricular injection of 6-hydroxydopamine a 59% decrease in cortical noradrenaline content was associated with a 44% decrease in [3H]purine release. Conversely, no change in [3H]purine release was found in rats in which a 89% decrease in cortical serotonin content was induced by intra-cerebroventricular injection of 5,7-dihydroxytryptamine. The decrease in [3H]purine release after the lesion of the cholinergic and noradrenergic pathways may depend on metabolic changes, a loss of a stimulating influence of acetylcholine and noradrenaline or may indicate a release of [3H]purine from cholinergic and noradrenergic fibres.

Effects of novelty and habituation on acetylcholine, GABA, and glutamate release from the frontal cortex and hippocampus of freely moving rats.

The involvement of the forebrain cholinergic system in arousal, learning and memory has been well established. Other neurotransmitters such as GABA and glutamate may be involved in the mechanisms of memory by modulating the forebrain cholinergic pathways. We studied the activity of cortical and hippocampal cholinergic, GABAergic and glutamatergic systems during novelty and habituation in the rat using microdialysis. After establishing basal release of the neurotransmitters, the animals were transferred to a novel environment and allowed to explore it twice consecutively for 30 min (60 min apart; exploration I and II). The motor activity was monitored. Samples were collected throughout the experiment and the release of acetylcholine (ACh), GABA and glutamate was measured. During the two consecutive explorations of the arena, cortical and hippocampal, ACh release showed a significant tetrodotoxin-dependent increase which was higher during exploration I than II. The effect was more pronounced and longer-lasting in the hippocampus than in the cortex. Cortical GABA release increased significantly only during exploration II, while hippocampal GABA release did not increase during either exploration. Motor activity was higher during the first 10 min of exploration I and II and then gradually decreased during the further 20 min. Both cortical and hippocampal ACh release were positively correlated with motor activity during exploration II, but not during I. During exploration II, cortical GABA release was inversely correlated, while hippocampal GABA release was positively correlated to motor activity. No change in cortical and hippocampal glutamate release was observed. In summary, ACh released by the animal placed in a novel environment seems to have two components, one related to motor activity and one related to attention, anxiety and fear. This second component disappears in the familiar environment, where ACh release is directly related to motor activity. The negative relationship between cortical GABA levels and motor activity may indicate that cortical GABAergic activity is involved in habituation.

Age-related secretion of androstenedione, testosterone and dihydrotestosterone by the human testis.

PIP: To study the production of testosterone, dihydrotestosterone (DHT), and androstenedione by the human testis during advancing age, these substances were measured in the spermatic venous blood plasma of 38 17-80 year old men. Samples of blood from the spermatic veins were collected during the operative repair of inguinal hernias. Plasma concentrations were determined by radioimmunoassay after paper chromatography. Control experiments were done with added known amounts of these substances to steroid free plasma. It was found that in old age the testicular production of DHT decreases significantly as well as its concentration in peripheral venous plasma. Spermatic androstenedione is unchanged while testosterone is decreased in senesence. This finding suggests that the decreased Leydig cell function in old age may be partly due to an enzymatic defect in the testicular steroidogenesis pathway because androstenedione is a direct precursor of testosterone.^ieng

Adenosine triphosphate release by osmotic shock and hemoglobin A1C in diabetic subjects' erythrocytes.

We investigated the significance of adenosine triphosphate (ATP) release from diabetic subjects' red blood cells (RBCs) following osmotic shock (OS) and its possible relationship with hemoglobin A1C (HbA1C) and with the RBC membrane protein skeleton. RBCs from type I (insulin-dependent [IDDM]) and type II (non-insulin-dependent [NIDDM]) diabetic subjects and age- and sex-matched control subjects were submitted to OS using NaCl solutions (from 0.9% to 0.045% final concentration). ATP release values were determined by the bioluminescent method. For pattern study, they were expressed both as absolute values and as percentages (%) of ATP maximum release (at 0.045% NaCl solution). Twenty-seven IDDM and 25 NIDDM subjects and two control groups were investigated. ATP content in RBCs was 2.08 +/- 0.19 pmol/10(4) RBC in IDDM and 1.23 +/- 0.20 pmol/10(4) RBC in NIDDM subjects. The ATP content of IDDM subjects' RBCs was significantly higher than that of the corresponding control group. ATP release at 0.49% NaCI OS, both as absolute value and as percentage value, was significantly lower in both diabetic groups, and ATP% was inversely correlated with HbA1" (IDDM: r = -.489, P < .01; NIDDM: r = -.654, P < .01), suggesting a possible relationship between Hb glycation, RBC membrane protein skeleton glycation, and its influence on ATP release by OS. In conclusion, the proposed method seems useful for measuring RBC ATP content and, at the same time, for monitoring the leak effect of the RBC membrane before it bursts.

Type-2 somatostatin receptor mRNA levels in breast and colon cancer determined by a quantitative RT-PCR assay based on dual label fluorogenic probe and the TaqMan technology.

We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neuroblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measurement and its applicability to large groups of patients, we present here an original 'real-time' quantitative RT-PCR method, based on a dual-labeled fluorogenic probe and the TaqMan technology. By this method, we have measured sst2 mRNA expression in 24 breast cancer samples and 26 colon carcinomas as well as on the corresponding non-adjacent non-neoplastic tissue of the same patients. The proposed method has a dynamic range of 4 x 10(4) to 4 x 10(8) molecules of sst2 mRNA. The intra-assay precision of the test, evaluated as signal detection variability, was 2.4%. Accuracy, evaluated by the addition of standard RNA to unknown samples, provided a mean recovery of 98+/-2%. A significant correlation has been observed in a study performed in 24 neuroblastoma samples measured both with the proposed method and with a competitive RT-PCR assay (r=0.913, p<0.001). In our preliminary clinical study, no significant differences were observed in sst2 mRNA levels between normal and tumor specimens in both colorectal (normal tissue 5.1 x 10(7)+/-2.0 x 10(7) molecules/microg total RNA, cancer tissue 9.7 x 10(7)+/-4.2 x 10(7)) and breast tumors (normal tissue 5.5 x 10(8)+/-2.0 x 10(8), cancer tissue 4.4 x 10(8)+/-3,7 x 10(8)).However, in colorectal cancer, sst2 mRNA values of subjects with high circulating carcinoembryonic antigen (CEA) levels (>5 ng/ml) were statistically lower (2.3 x 10(7)+/-6.2 x 10(6) molecules/, microg total RNA; p<0.05) than those of subjects with low CEA concentration (1.4 x 10(8)+/-6.7 x 10(7)). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T ratio) resulted significantly inversely related to CEA levels. In breast cancer, a significant difference was found between the mean N/T ratio of negative (below 10 fmol/mg protein) and positive estrogen receptor tumors (p<0.05). Analogous results were found selecting breast tumors on the basis of the progesterone receptor status (p<0.05). The proposed method is accurate, precise, sensitive and less labor-intensive than the competitive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it seems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens.