Stable-isotope dilution GC–MS approach for nitrite quantification in human whole blood,erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: Nitrite distribution in human blood

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC–MS using their ¹⁵N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC–MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0–4 °C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50 °C). Potassium ferricyanide (K₃Fe(CN)₆) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC–MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486 ± 280 nM in whole blood, 672 ± 496 nM in plasma (C_P), and 620 ± 350 nM in erythrocytes (C_E). The C_E-to-C_P ratio was 0.993 ± 0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC–MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.     

Consistency of the disposition index in the face of diet induced insulin resistance: potential role of FFA

Objective

Insulin resistance induces hyperinsulinemic compensation, which in turn maintains almost a constant disposition index. However, the signal that gives rise to the hyperinsulinemic compensation for insulin resistance remains unknown.

Methods

In a dog model of obesity we examined the possibility that potential early-week changes in plasma FFA, glucose, or both could be part of a cascade of signals that lead to compensatory hyperinsulinemia induced by insulin resistance.

Results

Hypercaloric high fat feeding in dogs resulted in modest weight gain, and an increase in adipose tissue with no change in the non-adipose tissue size. To compensate for the drop in insulin sensitivity, there was a significant rise in plasma insulin, which can be attributed in part to a decrease in the metabolic clearance rate of insulin and increased insulin secretion. In this study we observed complete compensation for high fat diet induced insulin resistance as measured by the disposition index. The compensatory hyperinsulinemia was coupled with significant changes in plasma FFAs and no change in plasma glucose.

Conclusions

We postulate that early in the development of diet induced insulin resistance, a change in plasma FFAs may directly, through signaling at the level of β-cell, or indirectly, by decreasing hepatic insulin clearance, result in the observed hyperinsulinemic compensation.     

Relationship between psychopathological factors and metabolic control in children and adolescents with insulin-dependent diabetes mellitus

This paper provides a critical overview of the literature on the relationship between psychological/psychopathological factors and metabolic control in children and adolescents with insulin dependent diabetes mellitus (IDDM). We discuss studies on individual and family psychopathological factors, as well as reports on the effects of psychoeducational/psychotherapeutic interventions on glycemic control in patients with IDDM aged <18 years. The analysis of the literature indicates that while evidence on the relationship between individual factors and metabolic control is still mixed, in part due to methodological issues, results from family studies do suggest that patients in dysfunctional families and children of parents with high degrees of psychopathology present with poor glycemic control. As for the effects of psychoeducational/psychotherapeutic interventions, limited but increasing evidence shows that they can actually contribute to improve metabolic control. We finally suggest some future underexplored avenues of research in the field, including studies on the psychopathological and neurobiological mechanisms underlying the above mentioned findings. All this body of research should provide a strong empirical rationale for allocating resources in order to include psychiatrists within the interdisciplinary diabetes health care team.     

Histomorphometric features of ventral prostate in different aged rats after central ghrelin treatment

Ghrelin, the endogenous ligand of growth hormone secretagogue receptor type 1a (GHS-R1a), has emerged as pleiotropic modulator of diverse biological functions, including energy homeostasis and recently, reproduction. The influence of intracerebroventricularly (ICV) administered ghrelin (1 μg/day/rat for 5 days) to rats of different ages, i.e, peripubertal (38 days), adult (60 days) and middle-aged (180 days) on the ventral prostate size and morphology, serum testosterone levels and testis weight was examined. Ghrelin treatment significantly increased (p < 0.05) absolute ventral prostate weight in peripubertal and middle-aged rats, by 27% and 37% respectively, due to enhancement of epithelial and/or luminal compartment of the gland. In adult rats, both absolute and relative volumes of the acinar lumen were significantly decreased (p < 0.05), by 38% and 44% respectively, which was associated with significant increases (p < 0.05) in relative and absolute volumes of interacinar stroma, whereas ventral prostate weigh was unchanged. Irrespective of animal age, ghrelin did not affect serum testosterone levels. These are the first results of ghrelin treatment effects on healthy prostate appearance, which allow us to conclude that the rat ventral prostate response to ghrelin depends on the developmental stage of animals. Our results merit further investigations and may have clinical implications, especially in the light of data on possible role of ghrelin in prostate hypertrophy and adenomas.     

Causal connection of non-specific low back pain and disc degeneration in children with transitional vertebra and/or Spina bifida occulta: role of magnetic resonance--prospective study

The problem of low back pain (LBP) in children is very common and many specialists are dealing with it in everyday practice. The cause for low back pain often is not found and classified under the diagnosis of non specific low back pain. The objective of this prospective study is to determine wether children with non specific low back pain and existence of anomalies in LS spine (transitional vertebra- TV and/or Spina bifida occulta SBO) also have the degeneration of the intervertebral disc (DD) L4-L5 and/or L5-S1. This prospective study included 69 patients from 8 to 16 years of age (X 12.81) of whom 40 were male (57.97%), and 29 female (42.03%). They all were examinated in University of Zagreb, "Sestre milosrdnice" University Hospital Center, Zagreb Children's Hospital, Department of Orthopaedic, Zagreb, Croatia. The reason of their visit was non specific low back pain. Pain was measured by visual analog scale (VAS) and mean score was three, duration of pain was between two and four weeks. Also, pain was sporadic, during daytime and not connected with level of physical activity. They all have undergone an algorithm of radiological examinations. Standard AP and LL radiographs (RTG) were made, as well as magnetic resonance (MR) of LS spine and sacrum in sagittal and transversal plane in T1 and T2 weighted sequence. The anomalies of L5 and S1 were found in 65 patients: transitional vertebra classified according to Castellvi et al. and SBO. In MRI in T2 weighted sequence DD was found in 61 patients which was classified modified from Pearce. Data analysis and comparison showed that 56patients with TV and/or SBO have changes on vertebral dynamic segment L5-S1 (VDS) and that means DD. In 13 patients only DD or spinal anomaly (TV and/or SBO) were found. Correlation between anomalies and DD in those patients was established by McNemar analysis and has shown significant difference (p=0.581) in favour of the patients with anomaly and DD. This has established that all of 56 patients with spinal anomaly could have DD as known cause of LBP.     

Residues of lindane in adipose tissue of lambs and sheep from islands Krk and Cres

Concentrations of lindane from adipose tissue i.e. omentum of sheep from homesteads of island Krk that were treated with lindane, were compared with the concentrations of lindane measured in adipose tissue of non treated sheep from husbandry cooperative on island Cres. Evaluation of food safety for this samples were estimated by comparing obtained results and maximum tolerance concentration (MTC) for lindane (2.0 and 0.2 mg/kg). In treated lambs from Krk measured concentrations of lindane were in range from 0.0038 to 1.8644 mg/kg, and in treated sheep from 0.0094 to 1.646 mg/kg. In control group of lambs and sheep concentration of lindane was < or = 0.0051 mg/kg. Estimation of food safety based on the results from this research are discussable, regarding the fact that MTC prescribed before year 2007 was 2.0 mg/kg. When the new law Regulations of MTC for residues of pesticides in food and animal feed (NN 119/07) came into the force prescribed MTC was 0.02 mg/kg. If food safety of sheep meat from Krk would be estimated based on latter, then 73.4% of samples of lamb meat and 80% of sheep meat could be proclaimed unsafe for human consumption. It is encouraging that after completion of using lindane in year 2005, levels of lindane in adipose tissue of lamb from Krk in year 2006 were under the concentration set by new legislate (< 0.02 mg/kg).     

Geochemical and petrographic evidence of meteoric diagenesis in tufa deposits in Northern Dalmatia (Zrmanja and Krupa Rivers, Croatia)

Summary Holocene tufa deposits in the Northern Dalmatia region of Croatia occur along the banks and at present sites of waterfalls of the Zrmanja river and its tributary the Krupa river. Petrographic, geochemical and statistical analyses have been used to relate textural features and trace element composition to the degree of meteoric diagenesis. Trace metal concentrations associated with carbonate phase were determined by sequential extraction procedure. The insoluble residue (I.R.) is predominantly of primary origin and its amount ranges from 3% to 29%, averaging 13.75%, thus permitting variable water/rock ratios of the stabilazing meteoric system. All the evidence points to a scarcity of post-depositional recrystallization and cementation, but where present these features are accompanied by I.R., Mg, Sr and Zn depletion and Fe enrichment.     

Zinc-decorated silica-coated magnetic nanoparticles for protein binding and controlled release

The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.     

Protection induced by Plasmodium falciparum MSP1(42) is strain-specific, antigen and adjuvant dependent, and correlates with antibody responses

Vaccination with Plasmodium falciparum MSP1(42)/complete Freund's adjuvant (FA) followed by MSP1(42)/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP1(42) expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP1(42) in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = -0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = -0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP1(42) vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component.