On the Use of Buffy or Whole Blood for Obtaining DNA of High Quality and Functionality: What Is the Best Option?

Human biobanks are collections of biological samples and health information that allow the organization of biomedical research for upgrading the knowledge of human disorders from different diseases (cancer, allergies, rare diseases, etc.), and reach real answers for diagnosis and treatment. A wide range of samples can be stored in these biorepositories such as hair, nails, urine, tissue, whole blood, red blood cells, buffy coat, plasma, serum, DNA, and RNA. Among these, buffy coat and whole blood are widely used by researchers because they can obtain DNA and RNA from these matrices. Some preliminary studies have been performed on animals to evaluate the quality and functionality of the nucleic acids obtained from some of these matrices, although more in-depth studies are needed in this area. In this study, blood samples extracted by venipuncture from 30 healthy volunteers were used to obtain DNA from buffy coat and whole blood. The purity and integrity of the nucleic acids obtained were assessed by spectrophotometry, fluorimetry, and agarose electrophoresis, and functionality was assessed by PCR and real-time PCR. Another aspect tested in this study was based on the comparison between short-term and long-term storage at -80°C and fresh samples from both matrices to evaluate the storage conditions at the biobank. Results showed differences in the yield obtained from both matrices as a function of the storage time, although the functionality of all the obtained DNA remained intact.

Analysis of the Spanish CCR5-∆32 inventory of cord blood units: lower cell counts in homozygous donors.

The possibility to use CCR5-∆32 umbilical cord blood to cure HIV infection in patients in need of a hematopoietic transplant has been suggested. The less stringent HLA compatibility needed in this type of transplant facilitates the search of a suitable donor having the CCR5-∆32 mutation. To achieve an inventory of CCR5-∆32 cord blood units, the 20,236 best cell quality units of the Spanish Registry were genotyped. Furthermore, their CD34+ and total nucleated cells counts, blood type, gender, HLA and donor's geographical and ancestral origin were analyzed. The results showed 130 (0.64%) units homozygous for the deletion, 2,646 (13.08%) heterozygous and 17,460 (86.28%) did not present the mutation. Interestingly, a significant lower amount of CD34+ cells was found in the CCR5-∆32 homozygous units. In addition, a significant association was found among donor's ancestral origin and the mutation, with a higher percentage of CCR5-∆32 units with a European ancestry. In summary, identification of a relatively high number of CCR5-∆32 units is feasible and will facilitate the development of clinical trials for HIV cure in patients requiring hematopoietic transplantation. Further studies are required to understand the significance of lower cell counts within the CCR5-∆32 homozygous group and its clinical impact.

Dietary fatty acids modulate adipocyte TNFa production via regulation of its DNA promoter methylation levels.

The factors regulating TNF alpha (TNFa) levels could be considered therapeutic targets against metabolic syndrome development. DNA methylation is a potent regulator of gene expression and may be associated with protein levels. In this study we investigate whether the effect of dietary fatty acids on TNFa released from adipocytes might be associated with modifications of the TNFa promoter DNA methylation status. A group of rats was assigned to three diets with a different composition of saturated, monounsaturated and polyunsaturated fatty acids. Samples of visceral adipose tissues were taken for adipocyte isolation, in which released TNFa levels were measured, and for methylation and expression studies. In addition, 3 T3-L1 cells were treated with palmitic, oleic and linoleic acids, with and without 5-Azacitydine (5-AZA). After treatments, cells and supernatants were included in the same analyses as rat samples. TNFa promoter methylation levels, gene expression and secretion were different according to the diets and fatty acid treatments associated with them. Cells treated with 5-AZA displayed higher TNFa levels than in the absence of 5-AZA, without differences between fatty acids. According to our results, dietary fatty acid regulation of adipocyte TNFa levels may be mediated by epigenetic modifications of the TNFa promoter DNA methylation levels.

Methylation patterns of Vegfb promoter are associated with gene and protein expression levels: the effects of dietary fatty acids.

PURPOSE: We have investigated the epigenetic regulation by dietary fatty acids of Vegfb levels in rats' white adipose tissue and 3T3-L1 cells. METHODS: A group of rats were assigned to three diets, each one with a different composition of saturated, monounsaturated and polyunsaturated fatty acids. Samples of white adipose tissues were taken for the methylation and expression studies. Additionally, 3T3-L1 cells were treated with palmitic, oleic, and linoleic fatty acids. After treatment, cells were harvested and genetic material was extracted for the analysis of Vegfb levels. RESULTS: We report evidence of changes in the methylation levels of the CpG island at the Vegfb promoter and in the Vegfb expression levels in vivo and in vitro by dietary fatty acid, with the main contribution of the linoleic fatty acid. Vegfb promoter methylation levels were closely related to the Vegfb gene expression. CONCLUSION: According to our results, the regulation of Vegfb gene expression by dietary fatty acids may be mediated, at least in part, by epigenetic modifications on Vegfb promoter methylation. Considering the deep association between angiogenesis and tissue growth, we suggest the nutriepigenetic regulation of Vegfb as a key target in the control of the adipose tissue expansion.

Adipose tissue characteristics related to weight z-score in childhood.

BACKGROUND: Childhood obesity has grown very fast over recent decades and now it represents a serious public health problem. The number of adipocytes is set in childhood and adolescence and then, an effective understanding of the development of adipose tissue during these periods will help in the prevention of this pathology. OBJECTIVES: The current study aimed to determine which adipose tissue characteristics are related to a high weight Z-score in childhood. PATIENTS AND METHODS: The current study included 82 children aged 5-130 months who underwent inguinal hernia surgery. Anthropometric variables were measured, and a nutritional and physical activity questionnaire was completed. Subcutaneous adipose tissue samples, taken during the operation, were analyzed for preadipocyte number, adipocyte volume, fatty acid composition (gas chromatography of FAME), and relative gene expression of various genes (real time PCR). RESULTS: The results showed that children with a higher weight Z-score spend more time in sedentary activities and less time running or involved in active games. SCD-1 activity index, arachidonic/linoleic index, and adipocyte volume were significantly higher in children with a weight Z-score greater than 0. The preadipocyte number and the genetic expression of the studied genes did not differ between the groups. A multiple regression analysis was done to determine which variables were related to the weight Z-score. R2 values indicated that the model which included adipocyte volume, SREBP-1c, SCD-1 expression, and activity index, predicted 59% of the variability in the weight Z-score among the children. The main variables associated with adipocyte volume were PPARγ, Adiponectin, CB1R expressions, as well as the SCD-1 activity and normalized weight. CONCLUSIONS: It was concluded that in childhood, the weight Z-score is related to adipocyte volume and adipose tissue gene expression.

High resolution melting curve analysis of DNA samples isolated by different DNA extraction methods.

BACKGROUND: High resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. Melting of double-stranded DNA molecules is influenced by several factors. We evaluated the influence of the DNA isolation method in the melting curve analysis to detect genetic variations. METHODS: We isolated DNA from whole blood of 547 subjects by two different methods: Maxwell 16 Instrument and DNA FlexiGene Kit. A fragment of 159 bp was amplified and analyzed by high resolution melting. Those samples that showed a different melting curve pattern were sequenced. RESULTS: Of the samples extracted with the Maxwell 16 Instrument, 42% showed variation compared with 0.18% of the samples extracted with DNA FlexiGene Kit. After sequencing, we showed that all samples extracted with the Maxwell 16 Instrument were false positive except one, which coincided with the only sample that showed variation in those extracted with the DNA FlexiGene Kit. CONCLUSION: The method used to extract DNA is an important factor to consider in the analysis of melting curves obtained by high resolution melting, as it may influence the melting behaviour of the samples, giving false positive results in the detection of genetic variants.

Effect of insulin analogues on 3t3-l1 adipogenesis and lipolysis.

BACKGROUND: Insulin has several biological functions besides glycaemic control. We investigated and compared the effects of six different commercial insulins on adipocyte cell differentiation, the lipolytic activity of differentiated cells, and the expression levels of genes involved in adipogenesis and associated with insulin activity. MATERIALS AND METHODS: 3T3-L1 cells were induced to differentiate with six commercial insulins: glargine, lispro, aspart, detemir, NPH and regular recombinant human insulin (used as control). Cell differentiation, lipolysis and gene expression were measured at day 7 (D7) and day 10 (D10) after induction of differentiation in these cells. RESULTS: The highest values of cell differentiation and lipolysis were found at D10 for all the insulins used. Preadipocyte differentiation differed at both times depending on the insulin used, with detemir insulin being the least adipogenic. The PPARγ mRNA level varied according to the insulin and was a good genetic marker of adipogenesis at D7. Cells treated with glargine insulin showed the highest lipolysis and HSL expression on both days. Gene expression levels of InsR, SREBP-1c and SCD-1 differed depending on the insulin studied. CONCLUSIONS: Detemir insulin was the least adipogenic of the insulins tested, whereas treatment with glargine insulin tended to produce the highest lipolysis levels. Under these experimental conditions, the modifications made in commercial insulins to improve glycaemic control also affect adipocyte differentiation, the lipolysis level of differentiated cells, and the expression of different genes that can modify metabolic pathways independently of glucose metabolism.

The -30G>A polymorphism of the glucokinase gene promoter is associated with obesity in a population from southern Spain.

The aim of this study was to test the hypothesis of an association between the -30G>A polymorphism of the promoter of the glucokinase gene and the prevalence and incidence of obesity. We studied the -30G>A polymorphism of the glucokinase gene promoter in 981 persons, of whom 866 were seen again 6 years later. All the persons underwent an oral glucose-tolerance test and the BMI (weight/height(2)) was recorded. The -30G>A polymorphism of the glucokinase gene promoter was studied using RFLP-PCR. At the initial study, the probability of having a BMI > or =25 in carriers of the A allele was significantly lower than expected by chance (odds ratio (OR) = 0.63; 95% confidence interval (CI) = 0.456-0.885). In those persons with a BMI > or =30 at the first study, the probability at 6 years of losing weight (reaching a BMI < 30) was greater in carriers of the A allele (OR = 0.22; 95% CI = 0.087-0.576). The increase in weight over these 6 years, taken as a continuous variable, was significantly less only in those persons who were originally obese (P = 0.018). In conclusion, in a population from southern Spain, carriers of the A allele of the -30G>A polymorphism in the promoter of the glucokinase gene had a lower risk for obesity and the likelihood of losing weight was greater in those obese persons who had the A allele (GA or AA).

Dietary oleic acid and adipocyte lipolytic activity in culture.

Although various studies have noted fatty-acid-mediated regulation of adipocyte lipolysis, determining the isolated effect of a single fatty acid is more difficult. We examined the influence of dietary oleic acid on adipose cell lipolytic activity and the tissue fat content independently of the variation in other dietary fatty acids. We fed 48 rats with six diets designed so that the oleic acid content was not correlated with the content of any other fatty acid and studied the lipolytic activity and fatty acid content of the tissues. There were no differences in the weight of the animals after the diet. The muscle fat content and the epinephrine-stimulated lipolytic activity varied significantly according to the dietary levels of oleic acid and the tissues, showing a dose-dependent behavior of the dietary oleic acid concentration. The results of this study show that diets rich in oleic acid have a beneficial effect on the regulation of lipid metabolism and body weight homeostasis.

Structure-function analysis of the alpha5 and the alpha13 helices of human glucokinase: description of two novel activating mutations.

It was recently described that the alpha5 and the alpha13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site-directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild-type and previously defined activating mutations in these helices (A456V and Y214C). We found two new activating mutations, A460R and Y215A, which increase the affinity of the enzyme for glucose. Our results suggest that substitutions in the alpha5 or the alpha13 helices that favor the closed, active conformation of the enzyme, either by improving the interaction with surrounding residues or by improving the flexibility of the region defined by these two helices, enhance the affinity of the enzyme for glucose, and therefore its performance as a glucose phosphorylating enzyme.

Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation.

Glucokinase (GK) is a glycolytic key enzyme that functions as a glucose sensor in the pancreatic beta-cell, where it governs glucose-stimulated insulin secretion (GSIS). Heterozygous inactivating mutations in the glucokinase gene (GCK) cause a mild form of diabetes (maturity-onset diabetes of the young [MODY]2), and activating mutations have been associated with a mild form of familial hyperinsulinemic hypoglycemia. We describe the first case of severe persistent hyperinsulinemic hypoglycemia due to a "de novo" mutation in GCK (Y214C). A baby girl presented with hypoglycemic seizures since the first postnatal day as well as with inappropriate hyperinsulinemia. Severe hypoglycemia persisted even after treatment with diazoxide and subtotal pancreatectomy, leading to irreversible brain damage. Pancreatic histology revealed abnormally large and hyperfunctional islets. The mutation is located in the putative allosteric activator domain of the protein. Functional studies of purified recombinant glutathionyl S-transferase fusion protein of GK-Y214C showed a sixfold increase in its affinity for glucose, a lowered cooperativity, and increased kcat. The relative activity index of GK-Y214C was 130, and the threshold for GSIS predicted by mathematical modeling was 0.8 mmol/l, compared with 5 mmol/l in the wild-type enzyme. In conclusion, we have identified a de novo GCK activating mutation that causes hyperinsulinemic hypoglycemia of exceptional severity. These findings demonstrate that the range of the clinical phenotype caused by GCK mutations varies from complete insulin deficiency to extreme hyperinsulinemia.

Hypertension is related to the degradation of dietary frying oils.

BACKGROUND: The family kitchen resembles an uncontrolled laboratory experiment, and some discrepancies in the relation between the risk of hypertension and dietary fat may be partly due to the manipulation to which the fats were subjected. OBJECTIVE: We investigated whether deterioration in the quality of the cooking oils in the family household contributes to the risk of high blood pressure. DESIGN: The study was cross-sectional. Anthropometric measurements were obtained for 1226 persons aged 18-65 y who were selected randomly from the municipal census of Pizarra, Spain. An oral-glucose-tolerance test was given to 1020 of these persons. Samples of the cooking oil being used were taken from the kitchens of a random subset of 538 persons. The concentrations of polar compounds and polymers were used as markers of the deterioration of the oils. The strength of association between variables was measured by calculating the odds ratio from logistic models. RESULTS: Hypertension was strongly associated with obesity and was influenced by sex, diabetes, and age. The presence of excess polar compounds in the cooking oil and the use of sunflower oil were related to the risk of hypertension, whereas the concentration of monounsaturated fatty acids in the serum phospholipids was negatively related to this risk. These associations remained after inclusion in the models of age, sex, obesity, and the presence of carbohydrate metabolism disorder. CONCLUSIONS: The risk of hypertension is positively and independently associated with the intake of cooking oil polar compounds and inversely related to blood concentrations of monounsaturated fatty acids.

[Physical activity and cardiovascular and metabolic risk factors in general population]. / Actividad física y factores de riesgo cardiovascular y metabólico en la población general.

BACKGROUND AND OBJECTIVE: Our purpose was to evaluate in a cross-section, populational study the association between daily physical activity and various metabolic and cardiovascular disease risk factors. PATIENTS AND METHOD: A total of 1226 randomly selected persons (aged 18-65 years) from a town in southeast Spain were evaluated for studying the association between the level of daily physical activity, both in and out of working hours, and the following variables: body mass index, plasma lipids, hypertension, obesity, altered baseline glycemia, and abnormal glucose tolerance. RESULTS: The intensity of the physical activity at work was related negatively with insulin resistance and levels of LDL cholesterol, and positively with HDL cholesterol. The probability of hypertension, altered baseline glycemia, and abnormal glucose tolerance was greater in those undertaking less daily physical activity. CONCLUSIONS: Daily physical activity, considered as part of the lifestyle, is related with the presence of components of the metabolic syndrome. An increase in daily physical activity should contribute to improve preventible cardiovascular risk factors.

Actividad física y factores de riesgo cardiovascular y metabólico en la población general / Physical activity and cardiovascular and metabolic risk factors in general population

FUNDAMENTO Y OBJETIVO: Evaluar en un estudio poblacional la asociación entre la actividad física diaria y diferentes factores de riesgo metabólico y cardiovascular. PACIENTES Y MÉTODO: En 1.226 personas (edad: 18-65 años) aleatoriamente seleccionadas en una población del sudeste español se ha evaluado la asociación entre el nivel de actividad física diaria laboral y extralaboral con las siguientes variables: índice de masa corporal, lípidos plasmáticos, presencia de hipertensión arterial, obesidad, glucemia basal alterada y tolerancia anormal de la glucosa. RESULTADOS: La intensidad de la actividad física en el trabajo se correlacionó de manera negativa con las concentraciones de colesterol ligado a lipoproteínas de baja densidad y positivamente con las de colesterol ligado a lipoproteínas de alta densidad. La probabilidad de presentar hipertensión arterial, glucemia basal alterada o tolerancia anormal de la glucosa fue mayor en aquellos que tuvieron una menor actividad física diaria. CONCLUSIONES: La actividad física diaria, considerada como uno de los componentes del estilo de vida, se asocia con la presencia de los componentes del síndrome metabólico. Un aumento de la actividad física diaria debe contribuir a mejorar los patrones favorables a la prevención de los factores de riesgo cardiovascular (AU)

Redistribution of abdominal fat after a period of food restriction in rats is related to the type of dietary fat.

The aim of the present experiment was to test the hypothesis that during refeeding a redistribution of intra-abdominal fat takes place and that both the recovery of weight and the redistribution of intra-abdominal fat are related to the type of dietary fat. The experimental study was carried out using male Sprague-Dawley rats. Three groups of animals were fed diets with three different fatty acid profiles. Each group contained two branches, one fed normally and the other fed initially with a 50 % energy reduction followed by refeeding ad libitum with the same isoenergetic diet as the control branch, giving a total of six treatments. Measurements were made of the final and incremental weight of the rat, weight of the intra-abdominal adipose tissue (total intra-abdominal, epididymal, omental and retroperitoneal adipose tissue weight), and feed efficacy (weight increment/metabolizable energy intake). Carcass, epididymal, omental, and muscle lipid contents, carcass protein and energy density were also measured. The results revealed that diets rich in fish oil or olive oil increase catch-up growth more than diets rich in saturated fats. During refeeding the lipid content in the adipose tissue increases while that of muscle tissue decreases. A diet rich in saturated fats induces a relative increase in the amount of intra-abdominal adipose tissue. The lipid content in adipose and muscle tissues and the distribution of intra-abdominal fat can all be modified by the type of dietary fat.

Prevalence of latent autoimmune diabetes of adults (LADA) in Southern Spain.

OBJECTIVE: To study the prevalence of diabetes mellitus and islet autoantibodies in an adult population from Southern Spain. RESEARCH AND METHODS: A cross-sectional study in Southern Spain of 1226 people, age 18-65 years. Clinical data were obtained and a blood sample taken to measure autoantibodies (glutamic acid decarboxylase antibodies (GADAb), tyrosine phosphatase antibodies (IA2Ab), and insulin antibodies (IAA)). An oral glucose tolerance test (OGTT) was also given to 982 of the subjects. RESULTS: The overall prevalence of diabetes mellitus according to the WHO 1979 criteria was 10.9% and according to the ADA 1997 criteria it was 14.7% (8.8% were unaware of their diabetes). The prevalence of impaired fasting glucose (IFG) was 12.4% and of impaired glucose tolerance (IGT) 11.5%. The prevalence of GADAb+ in the general population was 0.9% and in the diabetic population 3.7%. There were no significant differences between groups in the prevalence of IA2Ab or IAA (both were 0.8% in the general population). Of the three autoantibodies studied, only GADAb were significantly different in the diabetic population (P=0.0006). CONCLUSIONS: The prevalence of Type 2 diabetes and LADA are high in the south of Spain.