Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

BACKGROUND: Nucleic acids, RNA among them, are widely used in biomedicine and Biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. METHODS AND RESULTS: In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. DISCUSSION: QIAcube™ and semi-automated extraction methods were perceived as the best options because of their lower variability, good functionality, and lower cost (P < 0.001). These data contribute to facilitating researchers or research service platforms (Biobanks) in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

Measurement of yield and quality of DNA in human buffy coat is extraction method dependent.

During the last few years, an important element in the improvement of the molecular biology techniques has been the necessity for availability of high quality and functionality DNA. Several DNA extraction procedures with different results in both performance and quality, have been proposed. In this study our objective was to determine the most reliable extraction method that balances DNA quantity, and to assess the sample quantification of the fluorometric DNA quantification methods. For this, blood extracted by venopunction from 20 healthy volunteers was used to obtain DNA from buffy coat, and 4 commercial DNA extraction kits were assessed as well as two fluorometric DNA quantification methods with protocols of different complexity. Results suggest that manual methods achieve higher quality and larger yields of DNA. DNA purity obtained with the 4 extraction kits evaluated through the 260/280 and 260/230 ratio showed that the Qiacube kit fulfilled the criteria established in this work, followed very close by the Flexigene kit. On the other hand, the fluorometric DNA methods used in the samples quantification showed a higher variability when using QuantiFlour method, obtaining better results probably due to the simplicity of this protocol.

Four new HLA class I alleles, HLA-A*02:681, HLA-A*30:111, HLA-A*68:164 and HLA-B*35:01:46.

Four new HLA class I alleles, HLA-A*02:681, HLA-A*30:111, HLA-A*68:164 and HLA-B*35:01:46 were described in Spaniards.

The new HLA-B*50:51 allele was generated by intralocus recombination between B*50:01:01:02 and B*14:02:01.

The new B*50:51 allele was found in 3 Caucasians from Southern Spain.

Growth hormone-releasing hormone is produced by adipocytes and regulates lipolysis through growth hormone receptor.

BACKGROUND: Growth hormone-releasing hormone (GHRH) has a crucial role in growth hormone (GH) secretion, but little is known about its production by adipocytes and its involvement in adipocyte metabolism. OBJECTIVES: To determine whether GHRH and its receptor (GHRH-R) are present in human adipocytes and to study their levels in obesity. Also, to analyze the effects of GHRH on human adipocyte differentiation and lipolysis. METHODS: GHRH/GHRH-R and GH/GH-R mRNA expression levels were analyzed in human mature adipocytes from non-obese and morbidly obese subjects. Human mesenchymal stem cells (HMSC) were differentiated to adipocytes with GHRH (10-14-10-8 M). Adipocyte differentiation, lipolysis and gene expression were measured and the effect of GH-R silencing was determined. RESULTS: Mature adipocytes from morbidly obese subjects showed a higher expression of GHRH and GH-R, and a lower expression of GHRH-R and GH than non-obese subjects (P<0.05). A total of 10-14-10-10 M GHRH induced an inhibition of lipid accumulation and PPAR-γ expression (P<0.05), and an increase in glycerol release and HSL expression (P<0.05) in human differentiated adipocytes. A total of 10-12-10-8 M GHRH decreased GHRH-R expression in human differentiated adipocytes (P<0.05). A total of 10-10-10-8 M GHRH increased GH and GH-R expression in human differentiated adipocytes (P<0.05). The effects of GHRH at 10-10 M on adipocyte differentiation and lipolysis were blocked when GH-R expression was silenced. CONCLUSIONS: GHRH and GHRH-R are expressed in human adipocytes and are negatively associated. GHRH at low doses may exert an anti-obesity effect by inhibiting HMSC differentiation in adipocytes and by increasing adipocyte lipolysis in an autocrine or paracrine pathway. These effects are mediated by GH and GH-R.

HLA-A*30:99 shows a new amino acid position 156 within HLA-A*30 subtypes.

A novel HLA-A*30:99 allele was characterized in a Spanish individual.

Characterization of two novel HLA-A null alleles: A*11:210N and A*26:107N.

Two new HLA-A null alleles were characterized, A*11:210N and A*26:107N.

A new HLA-B allele, B*44:203, sequenced in a Spanish Caucasian cord blood unit.

HLA-B*44:203 shows one nucleotide difference to B*44:03:01 at codon 171 (TAC>CAC, Y171>H171).

A new HLA allele, HLA-B*08:108, described in two unrelated Spanish individuals.

HLA-B*08:108 shows one nucleotide difference regarding B*08:01:01 at codon 109 (CTC>TTC, L109>F109).

Thyroid hormone receptor alpha gene variants increase the risk of developing obesity and show gene-diet interactions.

OBJECTIVE: Thyroid hormone receptor-beta resistance has been associated with metabolic traits. THRA gene sequencing of an obese woman (index case) who presented as empirical thyroid hormone receptor-α (THRA) resistance, disclosed a polymorphism (rs12939700) in a critical region involved in TRα alternative processing. DESIGN AND SUBJECTS: THRA gene variants were evaluated in three independent europid populations (i) in two population cohorts at baseline (n=3417 and n=2265), 6 years later (n=2139) and (ii) in 4734 high cardiovascular risk subjects (HCVR, PREDIMED trial). RESULTS: The minor allele of the index case polymorphism (rs12939700), despite having a very low frequency (4%), was significantly associated with higher body mass index (BMI) (P=0.042) in HCVR subjects. A more frequent THRA polymorphism (rs1568400) was associated with higher BMI in subjects from the population (P=0.00008 and P=0.05) after adjusting for several confounders. Rs1568400 was also strongly associated with fasting triglycerides (P dominant=3.99 × 10(-5)). In the same sample, 6 years later, age and sex-adjusted risk of developing obesity was significantly increased in GG homozygotes (odds ratio 2.93 (95% confidence interval, 1.05-6.95)). In contrast, no association between rs1568400 and BMI was observed in HCVR subjects, in whom obesity was highly prevalent. This might be explained by the presence of an interaction (P <0.001) among the rs1568400 variant, BMI and saturated fat intake. Only when saturated fat intake was high (>24.5 g d(-1)), GG carriers showed a significantly higher BMI than A carriers after controlling for energy intake and physical activity. CONCLUSIONS: THRA gene polymorphisms are associated with obesity development. This is a novel observation linking the THRA locus to metabolic phenotypes.

Description of two novel HLA-B alleles, B*37:34 and B*44:152.

Two novel HLA-B alleles were characterized. HLA-B*37:34 shows two nucleotide differences regarding B*37:10 at codons 79 (CGC>CGG) and 80 (ACC>ATC), resulting in one amino acid replacement at position 80 (T>I). HLA-B*44:152 differs from B*44:02:01 in one nucleotide at codon 81 (GCG>ACG) giving rise to a leucine to threonine substitution at position 81.

Nutritional regulation of interleukin-6 release from adipocytes.

BACKGROUND AND AIMS: As interleukin-6 (IL-6) has an important role in general metabolism with high circulating levels in obesity and other associated diseases, the factors regulating its synthesis and release have been considered possible therapeutic targets and have recently been studied. We examined the influence of three different diets, each having a different fatty acid composition--saturated, monounsaturated or polyunsaturated (coconut oil, olive oil and sunflower oil diets), on IL-6 release from rat adipocytes, and the interaction between diet and other regulatory factors of IL-6 release, such as epinephrine. METHODS: A group of rats was assigned to one of the three different diets, each with a significantly different concentration of saturated, monounsaturated and polyunsaturated fatty acids. Samples were taken from the omental adipose tissue for measurement of the triacylglycerol fatty acid composition of the tissues and for adipocyte isolation. IL-6 release from adipocytes was measured in vitro, under nonstimulated conditions and also with two concentrations of epinephrine in the medium. RESULTS: Animals fed with the olive oil diet showed lower values of IL-6 release with and without epinephrine stimulation. IL-6 release from adipocytes varied according to the diet, but not according to epinephrine dose. However, a significant interaction was found between the epinephrine dose and the diet in IL-6 release regulation. CONCLUSIONS: IL-6 release from adipocytes was markedly regulated by the dietary fatty acid composition, even under epinephrine stimulation, with lower values of IL-6 release in the olive oil diet. The study also showed that epinephrine regulation of IL-6 release was related to the diet.

Anti-oxidized low-density lipoprotein antibody levels are associated with the development of type 2 diabetes mellitus.

BACKGROUND: Anti-oxidized low-density lipoprotein (LDL) antibodies are associated with the oxidative capacity of plasma, but whether they protect or promote diabetes is unknown. We undertook a prospective study to determine the predictive capacity of anti-oxidized LDL antibodies for the onset of type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: We selected 391 non-diabetic women aged 18-65 years. The subjects were classified as being normal (oral glucose test tolerance normal, OGTT-N), or having impaired glucose tolerance (IGT), impaired fasting glucose (IFG) or T2DM according to their baseline glucose levels and after an OGTT. The same subjects were studied six years later. The levels of anti-oxidized LDL antibodies were classified as above or below the 50th percentile. RESULTS: Of the women who were OGTT-N at the start of the study and who had anti-oxidized LDL antibody levels below the 50th percentile, only 65.1% were still OGTT-N after 6 years versus 79.5% of those who had anti-oxidized LDL antibody levels above the 50th percentile (P = 0.015). Women who had IGT or IFG at the start of the study whose anti-oxidized LDL antibody levels were below the 50th percentile had a relative risk of 9.79 (95% confidence interval, 1.40-68.45) of developing diabetes (P < 0.001). Logistic regression analysis showed that the variables predicting the development of a carbohydrate metabolism disorder in the women after 6 years were body mass index (P < 0.001) and the levels of anti-oxidized LDL antibodies (P = 0.042). CONCLUSIONS: Levels of anti-oxidized LDL antibodies are independent predictors for the development of T2DM in women.

Effect of long-term administration of cross-sex hormone therapy on serum and urinary uric acid in transsexual persons.

BACKGROUND: Transsexual persons afford a very suitable model to study the effect of sex steroids on uric acid metabolism. DESIGN: This was a prospective study to evaluate the uric acid levels and fractional excretion of uric acid (FEUA) in a cohort of 69 healthy transsexual persons, 22 male-to-female transsexuals (MFTs) and 47 female-to-male transsexuals (FMTs). The subjects were studied at baseline and 1 and 2 yr after starting cross-sex hormone treatment. RESULTS: The baseline levels of uric acid were higher in the MFT group. Compared with baseline, uric acid levels had fallen significantly after 1 yr of hormone therapy in the MFT group and had risen significantly in the FMT group. The baseline FEUA was greater in the FMT group. After 2 yr of cross-sex hormone therapy, the FEUA had increased in MFTs (P = 0.001) and fallen in FMTs (P = 0.004). In MFTs, the levels of uric acid at 2 yr were lower in those who had received higher doses of estrogens (P = 0.03), and the FEUA was higher (P = 0.04). The FEUA at 2 yr was associated with both the estrogen dose (P = 0.02) and the serum levels of estradiol-17beta (P =0.03). In MFTs, a correlation was found after 2 yr of therapy between the homeostasis model assessment of insulin resistance and the serum uric acid (r = 0.59; P = 0.01). CONCLUSIONS: Serum levels of uric acid and the FEUA are altered in transsexuals as a result of cross-sex hormone therapy. The results concerning the MFT group support the hypothesis that the lower levels of uric acid in women are due to estrogen-induced increases in FEUA.

Incidence of type 2 diabetes in southern Spain (Pizarra Study).

BACKGROUND: Few European studies have used an oral glucose tolerance test (OGTT) to examine the incidence of type 2 diabetes. We determined the incidence of impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and type 2 diabetes in a population from southern Spain. MATERIAL AND METHODS: A population-based cohort study was undertaken in Pizarra, Spain. Baseline data were recorded on age, sex, weight, height, waist and hip circumferences, and diabetes status for 1051 persons, of whom 910 were free of type 2 diabetes (at-risk sample). Of these, 714 completed the 6-year follow-up study. Body mass index, waist-to-hip ratio and weight increase since baseline were calculated. The homeostasis model assessment equations were used to estimate the indices of insulin resistance and beta-cell function. Each person received an OGTT at baseline and after 6 years. RESULTS: Type 2 diabetes developed in 81 people for a total of 4253 person-years, representing an incidence of 19.1 cases per 1000 person-years (95% confidence interval, 15.3-23.6). Age and the presence of obesity, central obesity and carbohydrate metabolism disorders [IFG (cut off = 100 mg dL(-1), capillary blood glucose level), IGT or both] at baseline were significant markers for the onset of type 2 diabetes during follow-up. After adjusting for these variables, multivariate analysis showed weight increase, waist-to-hip ratio and the indices of insulin resistance and beta-cell function were significantly associated with the risk for type 2 diabetes. CONCLUSIONS: The incidence of type 2 diabetes in a population from southern Spain is high. It is probably associated with the high prevalence of obesity and weight increase in this population.

Association between MspI polymorphism of the APO AI gene and Type 2 diabetes mellitus.

AIMS: Genes of the Apo AI/CIII/AIV cluster on chromosome 11 have been related to plasma lipid patterns. The close relationship between carbohydrate metabolism and lipid metabolism warrants investigation of the association between this cluster and Type 2 diabetes mellitus. We therefore examined the possible association between polymorphisms of this cluster and Type 2 diabetes mellitus as part of a study of the prevalence of diabetes and the metabolic syndrome in southern Spain. METHODS: A total of 1224 persons were selected randomly from the town of Pizarra in the province of Malaga, southern Spain. The sample errors for the prevalence of Type 2 diabetes mellitus and the three polymorphisms studied were all < or = 4%. All subjects underwent phenotyping after an oral glucose tolerance test (75 g) (WHO 1998 criteria) and the XmnI and MspI polymorphisms of Apo AI and the SstI polymorphism of Apo CIII were genotyped. RESULTS: Those subjects with the mutated AA genotype of the MspI polymorphism (-75 G-->A) of Apo AI had a greater risk of impaired glucose tolerance [odds ratio (OR) = 1.95, CI = 1.02-3.8, P = 0.05], Type 2 diabetes mellitus, both known (OR = 7.38, CI = 1.3-39.7, P = 0.02) and unknown (OR = 3.7, CI = 1.4-9.9, P = 0.009). This risk was independent of age, sex, obesity, triglyceride level, HDL cholesterol and pattern of insulin resistance. CONCLUSIONS: Pending confirmation in prospective studies, the AA genotype of the MspI polymorphism of the Apo AI gene, within the Apo A-I/C-III/A-IV cluster, seems to be a risk factor for Type 2 diabetes mellitus.

Patterns of insulin resistance in the general population of southeast Spain.

The aim of this study was to evaluate patterns of insulin resistance in the general population. The study was cross sectional. Clinical, anthropometric, and lipid measurements were made in 1226 persons aged 18-65 years. An oral glucose tolerance test (OGTT) was performed in 1020 subjects, with insulin levels determined at baseline and after 2 h. The homeostasis model assessment insulin resistance index (HOMA IR) and HOMA beta-cell function were calculated. Compared with subjects with normal glucose tolerance, the groups with abnormal OGTT had different baseline insulinemia, 2 h post OGTT insulinemia, HOMA IR and HOMA beta-cell indices. Serum insulin levels at baseline and 2 h after OGTT showed a characteristic pattern for each category of glucose tolerance, resulting from the different insulin responses. In the subjects with normal glucose tolerance, the pattern of the relationships between both types of serum insulin levels was exactly the same, so that it was possible to determine risk groups according to the ratio of baseline serum insulin/2 h insulin. HOMA IR and HOMA beta-cell were significantly associated with the risk of impaired fasting glucose, previously unknown diabetes mellitus, and known diabetes mellitus. These results support the rationale for introducing preventive measures against insulin resistance in the general population.

Association of biological markers of activity of systemic lupus erythematosus with levels of anti-oxidized low-density lipoprotein antibodies.

OBJECTIVES: To study the levels of anti-oxidized low-density lipoprotein (LDL) antibodies in patients with systemic lupus erythematosus (SLE) at two different points during the disease, and evaluate their relation with markers of SLE activity in serial blood samples. To investigate the correlations at two points in time between anti-oxidized LDL antibodies and anti-beta2-glycoprotein-I antibodies, leucocytes, immunoglobulin G, anti-deoxyribonucleic acid, complement 3, complement 4 and the disease activity index. METHODS: A total of 49 patients with SLE according to ACR criteria were studied at two points, 3 to 4 months apart, Time 1 and Time 2. RESULTS: There were ostensible changes in levels of anti-oxidized LDL antibodies between Times 1 and 2, which correlated significantly with disease activity markers. The association between levels of anti-oxidized LDL antibodies and complement system activation remained after multiple regression analysis with stepwise adjustment. CONCLUSIONS: Antibody levels against oxidized LDL vary with time and are closely related to the degree of SLE activity. There is an association between levels of autoantibodies to oxidized LDL and complement system activation.

Oleic acid from cooking oils is associated with lower insulin resistance in the general population (Pizarra study).

AIM: To evaluate the relation between type of dietary fatty acid and degree of insulin resistance. DESIGN: A cross-sectional study. METHODS: Anthropometrical data were measured in 538 subjects, aged 18-65 Years, selected randomly from the municipal census of Pizarra (Spain). An oral glucose tolerance test (OGTT) was given to all subjects and measurements were made of glycemia, insulinemia and the proportion of fatty acids in plasma phospholipids. Insulin resistance (IR) was estimated by homeostasis model assessment. Samples of cooking oil being used were obtained from the kitchens. The strength of association between variables was measured by calculating the odds ratio (OR) from logistic models, and the relationships were measured by linear correlation coefficients. RESULTS: Insulin resistance was significantly less in people who used olive oil compared with those who used sunflower oil or a mixture. Statistical significance remained in the group of people with normal OGTT after adjusting for obesity. In the whole sample, IR correlated negatively with the concentration of oleic acid (r=-0.11; P=0.02) and positively with that of linoleic acid (r=0.10; P=0.02) from the cooking oil. In subjects with normal OGTT, IR correlated negatively with oleic acid from cooking oil (r=-0.17; P=0.004) and from plasma phospholipids (r=-0.11; P=0.01) and positively with the concentration of linoleic acid in cooking oil (r=0.18; P=0.004) and plasma phospholipids (r=0.12; P=0.005). The risk (OR) of having raised IR was significantly lower in people who consumed olive oil, either alone (OR=0.50) or mixed (OR=0.52) compared with those who consumed only sunflower oil. CONCLUSION: There is an association between the intake of oleic acid, the composition of oleic acid in plasma phospholipids and peripheral insulin action.