Creatine in Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial

GUALANO, B., V. DE. SALLES PAINNELI, H. ROSCHEL, G. G. ARTIOLI, M. NEVES JR, A. L. DE SA PINTO, M. E. DA SILVA, M. R. CUNHA, M. C. G. OTADUY, C. DA COSTA LEITE, J. C. FERREIRA, R. M. PEREIRA, P. C. BRUM, E. BONFA, and A. H. LANCHA JR. Creatine in Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial. Med. Sci. Sports Exerc., Vol. 43, No. 5, pp. 770-778, 2011. Creatine supplementation improves glucose tolerance in healthy subjects. Purposes: The aim was to investigate whether creatine supplementation has a beneficial effect on glycemic control of type 2 diabetic patients undergoing exercise training. Methods: A 12-wk randomized, double-blind, placebo-controlled trial was performed. The patients were allocated to receive either creatine (CR) (5 g.d(-1)) or placebo (PL) and were enrolled in an exercise training program. The primary outcome was glycosylated hemoglobin (Hb(A1c)). Secondary outcomes included the area under the curve of glucose, insulin, and C-peptide and insulin sensitivity indexes. Physical capacity, lipid profile, and GLUT-4 protein expression and translocation were also assessed. Results: Twenty-five subjects were analyzed (CR: n = 13; PL: n = 12). Hb(A1c) was significantly reduced in the creatine group when compared with the placebo group (CR: PRE = 7.4 +/- 0.7, POST = 6.4 +/- 0.4; PL: PRE = 7.5 +/- 0.6, POST = 7.6 +/- 0.7; P = 0.004; difference = -1.1%, 95% confidence interval = -1.9% to -0.4%). The delta area under the curve of glucose concentration was significantly lower in the CR group than in the PL group (CR = -7790 +/- 4600, PL = 2008 +/- 7614; P = 0.05). The CR group also presented decreased glycemia at times 0, 30, and 60 min during a meal tolerance test and increased GLUT-4 translocation. Insulin and C-peptide concentrations, surrogates of insulin sensitivity, physical capacity, lipid profile, and adverse effects were comparable between the groups. Conclusions: Creatine supplementation combined with an exercise program improves glycemic control in type 2 diabetic patients. The underlying mechanism seems to be related to an increase in GLUT-4 recruitment to the sarcolemma.

Three-dimensional visualization of human hemoglobin phenotypes with HPLC

Hemoglobinopathies were included in the Brazilian Neonatal Screening Program on June 6, 2001. Automated high-performance liquid chromatography (HPLC) was indicated as one of the diagnostic methods. The amount of information generated by these systems is immense, and the behavior of groups cannot always be observed in individual analyses. Three-dimensional (3-D) visualization techniques can be applied to extract this information, for extracting patterns, trends or relations from the results stored in databases. We applied the 3-D visualization tool to analyze patterns in the results of hemoglobinopathy based on neonatal diagnosis by HPLC. The laboratory results of 2520 newborn analyses carried out in 2001 and 2002 were used. The ""Fast"", ""F1"", ""F"" and ""A"" peaks, which were detected by the analytical system, were chosen as attributes for mapping. To establish a behavior pattern, the results were classified into groups according to hemoglobin phenotype: normal (N = 2169), variant (N = 73) and thalassemia (N = 279). 3-D visualization was made with the FastMap DB tool; there were two distribution patterns in the normal group, due to variation in the amplitude of the values obtained by HPLC for the F1 window. It allowed separation of the samples with normal Hb from those with alpha thalassemia, based on a significant difference (P < 0.05) between the mean values of the ""Fast"" and ""A"" peaks, demonstrating the need for better evaluation of chromatograms; this method could be used to help diagnose alpha thalassemia in newborns.

Dynamic light scattering and optical absorption spectroscopy study of pH and temperature stabilities of the extracellular hemoglobin of Glossoscolex paulistus

The extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted of subunits containing heme groups, monomers and trimers, and nonheme structures, called linkers, and the whole protein has a minimum molecular mass near 3.1 x 10(6) Da. This and other proteins of the same family are useful model systems for developing blood substitutes due to their extracellular nature, large size, and resistance to oxidation. HbGp samples were studied by dynamic light scattering (DLS). In the pH range 6.0-8.0, HbGp is stable and has a monodisperse size distribution with a z-average hydrodynamic diameter (D-h) of 27 +/- 1 nm. A more alkaline pH induced an irreversible dissociation process, resulting in a smaller D-h of 10 +/- 1 nm. The decrease in D-h suggests a complete hemoglobin dissociation. Gel filtration chromatography was used to show unequivocally the oligomeric dissociation observed at alkaline pH. At pH 9.0, the dissociation kinetics is slow, taking a minimum of 24 h to be completed. Dissociation rate constants progressively increase at higher pH, becoming, at pH 10.5, not detectable by DILS. Protein temperature stability was also pH-dependent. Melting curves for HbGp showed oligomeric dissociation and protein denaturation as a function of pH. Dissociation temperatures were lower at higher pH. Kinetic studies were also performed using ultraviolet-visible absorption at the Soret band. Optical absorption monitors the hemoglobin autoxidation while DLS gives information regarding particle size changes in the process of protein dissociation. Absorption was analyzed at different pH values in the range 9.0-9.8 and at two temperatures, 25 degrees C and 38 degrees C. At 25 degrees C, for pH 9.0 and 9.3, the kinetics monitored by ultraviolet-visible absorption presents a monoexponential behavior, whereas for pH 9.6 and 9.8, a biexponential behavior was observed, consistent with heme heterogeneity at more alkaline pH. The kinetics at 38 degrees C is faster than that at 25 degrees C and is biexponential in the whole pH range. DLS dissociation rates are faster than the autoxidation dissociation rates at 25 degrees C. Autoxiclation and dissociation processes are intimately related, so that oligomeric protein dissociation promotes the increase of autoxidation rate and vice versa. The effect of dissociation is to change the kinetic character of the autoxidation of hemes from monoexponential to biexponential, whereas the reverse change is not as effective. This work shows that DLS can be used to follow, quantitatively and in real time, the kinetics of changes in the oligomerization of biologic complex supramolecular systems. Such information is relevant for the development of mimetic systems to be used as blood substitutes.

Hemoglobin Regeneration Efficiency in Anemic Rats: Effects on Bone Mineral Composition and Biomechanical Properties

This study reports the effects of dietary iron (Fe) deficiency and recovery on bone mineral composition and strength in anemic rats submitted to a hemoglobin (Hb) repletion assay. Weanling male Wistar rats were fed a low-Fe diet (12 mg/kg) for 15 days followed by 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as either ferrous sulfate (n = 8) or ferric pyrophosphate (FP; n = 12). At final day of each period (depletion and repletion), Fe-adequate animals were also euthanized. Iron status (blood Hb, Hb Fe pool, Hb regeneration efficiency), tibia mineral concentrations (Ca, Mg, Fe, Cu, and Zn) and biomechanical properties were evaluated. Iron-deficient rats had lower tibia Fe and Mg levels and bone strength when compared to controls. Yield load and resilience were positively related to tibia Mg levels (r = 0.47, P = 0.02 and r = 0.56, P = 0.004, respectively). Iron repletion did not recover tibia Mg concentrations impaired by Fe deficiency. Moreover, bone elastic properties were negatively affected by FP consumption. In conclusion, bone mineral composition and strength were affected by Fe deficiency, whereas dietary Fe source influenced tibia Mg and resistance in the period during which rats were recovering from anemia.

Novel and facile solution-phase synthesis of 2,5-diketopiperazines and O-glycosylated analogs

This work describes the synthesis in Solution of a series of related diketopiperazines with potential biological activities: cyclo(L-Pro-L-Ser), cyclo(L-Phe-L-Ser), cyclo(D-Phe-L-Ser) and the corresponding glycosylated analogs of the latter, cyclo[D-Phe-L-Ser(alpha GlcNAc)] and cyclo[D-Phe-L-Ser(beta GlcNAc)]. The synthetic approach involved coupling reactions of -OH or O-glycosylated serine benzyl esters with NFmoc-protected amino acids (Pro or Phe), followed by one-pot deprotection-cyclization reaction in the presence of 20% piperidine in DMF. (C) 2009 Elsevier Ltd. All rights reserved.

The Association of Hemoglobin A1c With Incident Heart Failure Among People Without Diabetes: The Atherosclerosis Risk in Communities Study

OBJECTIVE-This study sought to investigate an association of HbA1c (A1C) with incident heart failure among individuals without diabetes and compare it to fasting glucose. RESEARCH DESIGN AND METHODS-We studied 11,057 participants of the Atherosclerosis Risk in Communities (ARIC) Study without heart failure or diabetes at baseline and estimated hazard ratios of incident heart failure by categories of A1C (<5.0, 5.0-5.4 [reference], 5 5-59, and 6.0-6.4%) and fasting glucose (<90, 90-99 [reference], 100-109, and 110-125 mg/dl) using Cox proportional hazards models. RESULTS-A total of 841 cases of incident heart failure hospitalization or deaths (International Classification of Disease, 9th/10th Revision, 428/150) occurred during a median follow-up of 14.1 years (incidence rate 5.7 per 1,000 person-years). After the adjustment for covariates including fasting glucose, the hazard ratio of incident heart failure was higher in individuals with A1C 6.0-6.4% (1.40 [95% CI, 1 09-1.79]) and 5.5-6.0% (1.16 [0.98-1 37]) as compared with the reference group. Similar results were observed when adjusting for insulin level or limiting to heart failure cases without preceding coronary events or developed diabetes during follow-up. In contrast, elevated fasting glucose was not associated with heart failure after adjustment for covariates and A1C. Similar findings were observed when the top quartile (A1C, 5.7-6.4%, and fasting glucose, 108-125 mg/dl) was compared with the lowest quartile (<5 2% and <95 mg/dl, respectively). CONCLUSIONS-Elevated A1C (>= 5.5-6 0%) was associated with incident heart failure in a middle-aged population without diabetes, suggesting that chronic hyperglycemia prior to the development of diabetes contributes to development of heart failure. Diabetes 59:2020-2026, 2010

Distribution of Hemoglobin Phenotypes in Four Different Districts of Porto Velho, Rondonia, Brazil

Hemoglobin profile studies have been carried out in four samples from different districts of Porto Velho (Rondonia State) in the western Amazonian region of Brazil: Candelaria, Bate Estaca, Hemeron (at the State Blood Bank), and Sao Carlos. Samples from 337 unrelated individuals were collected during medical and paramedical team visits by professionals from the Instituto de Pesquisa em Patologia Tropical and the Centro de Pesquisa em Patologias Tropicais (both research institutes in tropical diseases). The aim of this study is to assess the frequency of alleles in the hemoglobin system, mainly alleles HB*A, *S, and *E. The overall phenotype frequencies were FIB A,S = 0.025, HB A,E = 0.006, and HB A,A = 0.969. Samples from the blood bank subjects and samples from the homogeneous areas of Silo Carlos and Candelaria plus Bate Estaca have a chi-square of heterogeneity of 6.383 (p = 0.041) and 8.406 (p = 0.015), respectively. The allele frequencies (HB*A = 0.984, HB*S = 0.012, and HB*E = 0.003) do not significantly differ from frequencies found in other Brazilian regions.

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P<0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO (P=0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women (P<0.01). We found significant positive correlations between pHb and pNOc (r=0.61; P<0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations (P=0.02; r=-0.32 and P=0.01: r=-0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations (P=0.03; r=-0.36 and P=0.01: r=-0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women. (C) 2010 Elsevier Inc. All rights reserved.

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved.

Diabetes and Vascular Disease: Basic Concepts of Nitric Oxide Physiology, Endothelial Dysfunction, Oxidative Stress and Therapeutic Possibilities

The vascular manifestations associated with diabetes mellitus (DM) result from the dysfunction of several vascular physiology components mainly involving the endothelium, vascular smooth muscle and platelets. It is also known that hyperglycemia-induced oxidative stress plays a role in the development of this dysfunction. This review considers the basic physiology of the endothelium, especially related to the synthesis and function of nitric oxide. We also discuss the pathophysiology of vascular disease associated with DM. This includes the role of hyperglycemia in the induction of oxidative stress and the role of advanced glycation end-products. We also consider therapeutic strategies.

Salivary Interleukin-6, Matrix Metalloproteinase-8, and Osteoprotegerin in Patients With Periodontitis and Diabetes

Background: Diabetes and periodontitis produce a protein discharge that can be reflected in saliva. This study evaluates the salivary concentrations of interleukin (IL)-6, matrix metalloproteinase (MMP)-8, and osteoprotegerin (OPG) in patients with periodontitis with type 2 diabetes. Methods: Whole saliva samples were obtained from 90 subjects who were divided into four groups: healthy (control; n = 22), untreated periodontitis (UPD; n = 24), diabetes mellitus (DM; n = 20), and UPD + DM (n = 24) groups. Clinical and metabolic data were recorded. Salivary IL-6, MMP-8, and OPG concentrations were determined by a standard enzyme-linked immunosorbent assay. Results: The UPD and UPD + DM groups exhibited higher salivary IL-6 than the control and DM groups (P <0.01). The salivary MMP-8 concentrations in all diseased groups (UPD, DM, and UPD + DM) were higher than in the control group (P <0.01). The salivary OPG concentrations in the DM group were higher than in the UPD and control groups (P<0.05). In the UPD + DM group, salivary IL-6 was correlated with glycated hemoglobin (HbA1c) levels (r = 0.60; P<0.05). The regression analysis indicated that the number of remaining teeth, clinical attachment level, and IL-6 might have influenced the HbA1c levels in patients with diabetes. Conclusions: Salivary 1L-6 concentrations were elevated in patients with periodontitis with or without diabetes. Salivary MMP-8 and OPG concentrations were elevated regardless of periodontal inflammation in patients with diabetes. Therefore, periodontitis and diabetes are conditions that may interfere with protein expression and should be considered when using saliva for diagnoses. J Periodontol 2010;81:384-391.

Hemopressins and other hemoglobin-derived peptides in mouse brain: comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice

P>Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpefat/fat mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpefat/fat mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is up-regulated in some but not all Cpefat/fat mouse brain regions.

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85 angstrom. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. (C) 2009 Elsevier Inc. All rights reserved.

Lipid hydroperoxide-induced and hemoglobin-enhanced oxidative damage to colon cancer cells

Epidemiological studies have indicated that Western diets are related to an increase in a series of malignancies. Among the compounds that are credited for this toxic effect are heme and lipid peroxides. We evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxides (LAOOH) on a series of toxicological endpoints, such as cytotoxicity, redox status, lipid peroxidation, and DNA damage. We demonstrated that the preincubation of SW480 cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, malonaldehyde formation, and DNA fragmentation and that these effects were related to the peroxide group and the heme present in Hb. Furthermore, Hb and LAOOH alone exerted a toxic effect on the endpoints assayed only at concentrations higher than 100 mu M. We were also able to show that SW480 cells presented a higher level of the modified DNA bases 8-oxo-7,8-dihydro-2`-deoxyguanosine and 1,N(2)-etheno-2`-deoxyguanosine compared to the control. Furthermore, incubations with Hb led to an increase in intracellular iron levels, and this high level of iron correlated with DNA oxidation, as measured as EndoIII- and Fpg-sensitive sites. Thus, Hb from either red meat or bowel bleeding could act as an enhancer of fatty acid hydroperoxide genotoxicity, which contributes to the accumulation of DNA lesions in colon cancer cells. (C) 2011 Elsevier Inc. All rights reserved.

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS): Effects of oligomeric dissociation

The present work focuses on the interaction between the zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp). Electronic optical absorption, fluorescence emission and circular dichroism spectroscopy techniques, together with Gel-filtration chromatography, were used in order to evaluate the oligomeric dissociation as well as the autoxidation of HbGp as a function of the interaction with HPS. A peculiar behavior was observed for the HPS-HbGp interaction: a complex ferric species formation equilibrium was promoted, as a consequence of the autoxidation and oligomeric dissociation processes. At pH 7.0, HPS is more effective up to 1 mM while at pH 9.0 the surfactant effect is more intense above 1 mM. Furthermore, the interaction of HPS with HbGp was clearly less intense than the interaction of this hemoglobin with cationic (CTAC) and anionic (SDS) surfactants. Probably, this lower interaction with HPS is due to two factors: (i) the lower electrostatic attraction between the HPS surfactant and the protein surface ionic sites when compared to the electrostatic interaction between HbGp and cationic and anionic surfactants, and (ii) the low cmc of HPS, which probably reduces the interaction of the surfactant in the monomeric form with the protein. The present work emphasizes the importance of the electrostatic contribution in the interaction between ionic surfactants and HbGp. Furthermore, in the whole HPS concentration range used in this study, no folding and autoxidation decrease induced by this surfactant were observed. This is quite different from the literature data on the interaction between surfactants and tetrameric hemoglobins, that supports the occurrence of this behavior for the intracellular hemoglobins at low surfactant concentration range. Spectroscopic data are discussed and compared with the literature in order to improve the understanding of hemoglobin-surfactant interaction as well as the acid isoelectric point (pI) influence of the giant extracellular hemoglobins on their structure-activity relationship. (c) 2007 Elsevier B.V. All rights reserved.