Adult Plasma Research Articles

The Effect of Age on the Glycosylation of Human Alpha-2-Macroglobulin

Introduction: Alpha-2-macroglobulin (a2m) is a plasma glycoprotein capable of inhibiting all classes of serine proteases. Within the coagulation and fibrinolytic systems, a2m inhibits several critical factors, including thrombin, plasmin, and activated protein C. Previous studies have shown that coagulation factor concentrations are highly variable with age. Notably, a2m levels are approximately twice as high in newborns compared to adults. Elevated levels of a2m in newborns may, in part, contribute to a resistance towards thrombotic events observed in this population. Protein glycosylation is known to affect protein activity as well as pharmacodynamics. The glycosylation profile of adult a2m has previously been analyzed and shown to contain 8 potential sites of N-linked glycosylation. Information regarding glycosylation of a2m in other age groups has yet to be elucidated. Therefore, the purpose of this study is to examine the differences in the glycosylation profiles between newborn and adult a2m.Methods: Normal adult pooled platelet-poor plasma was obtained commercially. Newborn platelet-poor plasma was isolated by standard methods from umbilical cord blood obtained immediately after delivery of healthy full-term infants. Pooled newborn plasma was constructed from 30 different donors. To evaluate the degree of N-linked glycosylation, plasma samples were enzymatically deglycosylated by peptide N-glycosidase F (PNGaseF). Samples were subjected to SDS polyacrylamide (5%) gel electrophoresis (SDS PAGE) and western blotting to detect a2m in plasma. To evaluate a2m sialic acid content, plasma samples were incubated with Neuraminidase from Clostridium perfringens. Following incubation, samples were subjected to native polyacrylamide (4%) gel electrophoresis and western blotting. To detect the presence of non-sialylated terminal galactose residues, plasma samples were incubated with immobilized Ricinus communis lectin, and lectin-bound molecules were separated from unbound molecules by centrifugation. Bound and unbound fractions were subjected to SDS-PAGE and western blotting as described above to detect a2m.Results: Deglycosylation of both newborn and adult a2m with PNGaseF resulted in a change in migration and apparent molecular weight on SDS-PAGE. There was no significant difference (p=0.28, n=3) between the change in apparent molecular weight for newborn a2m (16.1 ± 0.42 kDa) versus adult a2m (14.5 ± 0.99 kDa). On native polyacrylamide gel electrophoresis, newborn a2m exhibited slightly increased migration compared to adult a2m. This difference in migration was abolished following treatment with neuraminidase, indicating the variation in migration was due to differing sialic acid content. Additionally, a lower proportion of newborn a2m was bound to Ricinus communis compared to adult a2m. This indicates newborn a2m has fewer non-sialylated galactose residues available for binding to this lectin.Conclusions: To our knowledge, this is the first study investigating potential glycan heterogeneity between newborn and adult a2m molecules. The results from PNGaseF analyses indicate that there is no macroheterogeneity in total N-glycan content apparent between newborn and adult a2m. However, the observed increase in sialic acid content of newborn a2m compared to adult a2m may be responsible for age-related differences in pharmacodynamic and pharmacokinetic properties of this important protease inhibitor. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Plasmin Generation in Plasma By Heparin, Low Molecular Weight Heparin and a Covalent Antithrombin-Heparin Complex (ATH)

Introduction: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are widely used anticoagulants for thrombosis treatment. However, these anticoagulants have limitations such as increased bleeding, variable dose response, required frequent monitoring, and, in the case of LMWH, inability to inhibit thrombin. This has led to the development of a covalent complex of antithrombin and heparin (ATH), which has been shown to overcome many of these shortcomings. ATH has faster rates of inhibition of many coagulation factors, is able to inhibit clot-bound thrombin, and is a more effective inhibitor of both venous and arterial thrombosis in animal models. Moreover, in a rabbit thrombosis model, ATH has been shown to decrease clot mass and fibrin accretion, while the contrary was observed for UFH. From these observations, it was suggested that ATH may enhance fibrin breakdown and thus led to investigations into the effects of UFH and ATH on fibrinolysis. In vitro studies have shown that UFH enhances antithrombin inhibition of plasmin. In addition, ATH displays a slightly greater inhibition of plasmin generation and activity. Such studies were conducted in purified systems, in the absence of other plasmin inhibitors naturally present in plasma. Therefore, the aim of the present study was to compare the effects of UFH, LMWH, and ATH on plasmin generation in plasma.Methods: At 37°C tissue plasminogen activator (tPA) and soluble fibrin fragments (fib) were added to normal adult pooled platelet poor plasma supplemented with 0.35, 0.7, 1.4, or 2.1 U anti-Xa/ml UFH, LMWH, or ATH, to initiate plasmin generation (8.93nM tPA and 300µg/ml fib). At various time points, subsamples were mixed with excess plasminogen activator inhibitor 1 (PAI-1) (55.12nM) to stop further plasmin generation. The plasmin concentration at each time point was determined using a plasmin-specific chromogenic substrate and a standard curve produced from purified plasmin.Results: Comparisons of mean area under the curve (AUC) for plasmin generation displayed a significant decrease in plasmin generation in the presence of all three anticoagulants at all doses tested (p<0.05). Comparing the anticoagulants at similar doses, plasmin generation was significantly decreased in the presence of ATH (15384.66±1930.23nM/min) compared to LMWH (23892.28±3090.54nM/min) at 0.7 U/ml (p<0.05). At a dose of 1.4 U/ml, there was significantly less plasmin generated, over time, in the presence of UFH (20089.49±3022.1623nM/min) and ATH (19273.86±1805.7323nM/min) when compared to LMWH (24743.18±1265.1023nM/min) (p<0.05). There was no significant difference in plasmin inhibition between UFH and ATH at any of the doses tested.Conclusion: The present study supports previous findings that UFH and ATH can facilitate antithrombin inhibition of plasmin. It is also observed that LMWH catalyzes the inhibition of plasmin by antithrombin but possibly to a lesser extent. These findings suggest that ATH has a similar inhibitory effect on plasmin generation and activity in plasma compared to UFH, despite its overall superior anticoagulant properties. Therefore, previous in vivo observations displaying decrease in clot mass with administration of ATH was due to its enhanced anticoagulant abilities and not fibrinolysis enhancement. These findings add to our understanding of ATH mechanisms of action and aid in its development for clinical use. DisclosuresNo relevant conflicts of interest to declare.

Insulin glargine metabolite 21A-Gly-human insulin (M1) is the principal component circulating in the plasma of young children with type 1 diabetes: results from the PRESCHOOL study

Insulin glargine metabolite 21(A) -Gly-human insulin (M1) is the principal component circulating in plasma of adults with type 1 diabetes. The objective of this study was to confirm this finding in young children and to rule out accumulation of parent insulin glargine. Children with type 1 diabetes from the PRESCHOOL study, aged 2-6 yr, were treated with insulin glargine for 24 wk (n = 62). Blood samples were drawn at weeks 1, 2, and 4 approximately 24 h after the last dose and analyzed for glargine, M1, and Thr(30B) -des-M1 (M2) using immunoaffinity purification and liquid chromatography with mass spectrometry. The lower limit of quantification was 33 pmol/L for all analytes. M1 was the principal active component circulating in plasma. Mean (SD) plasma Ctrough values were 101 (138), 80 (122), and 79 (102) pmol/L following glargine doses of 0.33 (0.02), 0.34 (0.02), and 0.38 (0.03) U/kg at weeks 1, 2, and 4, respectively. Parent insulin glargine and M2 concentrations were below the level of quantification. These results are in line with those observed in adults and indicate no accumulation of the parent compound in this patient population. In young children with type 1 diabetes, the principal component circulating in plasma after subcutaneous injection of insulin glargine is M1, the pharmacologically active component. No accumulation of the parent insulin glargine was observed. These data provide additional evidence on the safety profile of insulin glargine in young children (Clinical trial identifier: NCT00993473).

Gender and age differences in growth factor concentrations from platelet-rich plasma in adults.

The use of platelet-rich plasma (PRP) to facilitate healing of orthopedic-related injuries has gained popularity; however, the clinical benefits are not consistent. Differences may result from variations in growth factor (GF) levels in normal populations. The purpose of this study was to determine if GF levels present in activated PRP preparations differed by gender and age (≤ 25 versus >25 years) in a healthy population (N = 102). All GFs analyzed (epidermal growth factor [EGF], hepatocyte growth factor [HGF], insulin growth factor-1 [IGF-1], platelet-derived growth factor-AB [PDGF-AB], platelet-derived growth factor-BB [PDGF-BB], transforming growth factor beta-1 [TGFβ-1], and vascular endothelial growth factor) had higher levels for females and for those ≤ 25 years old. Of the GFs tested, four of seven were significantly higher (p < 0.05) for females (EGF, HGF, IGF-1, PDGF-BB), the most significant being IGF-1 (female, 85.0; male, 69.3 ng/mL; p < 0.01). Five of seven GFs achieved significance (p < 0.05) for people ≤ 25 years old (EGF, IGF-1, PDGP-AB, PDGF-BB, and TGFβ-1), with IGF and PDGF-AB achieving p < 0.001 (≤ 25 years, 85.1; >25 years, 56.8, and ≤ 25 years, 7.66; >25 years, 5.77 ng/mL, respectively). Finally, for both genders, most of the GFs were positively correlated with all GFs. This study demonstrated that both age and gender account for variations in specific GFs present in PRP, and this may partially explain some of the inconsistent results of PRP clinical trials.

Patterns of within-clutch variation in yolk lutein in the Yellow-legged Gull Larus michahellis: the effects of egg laying order and laying date

In gull species, the level of carotenoids in egg yolks is known to decrease along the egg laying sequence. However, the extent of this laying order effect is expected to vary among clutches according to laying date, due to possible differences between early-nesting and late-nesting females with regards to their ability to control the transfer of maternal resources to the eggs. A laying date effect could also result from a seasonal decline in the availability of dietary carotenoids. In this study, we collected data from Yellow-legged Gulls (Larus michahellis) breeding in southern Tunisia to investigate how nesting date interacts with egg-laying order in shaping patterns of within-clutch variation in yolk lutein, the most abundant carotenoid in this species. In particular, we examined (1) whether yolk lutein concentration declined along the laying sequence and (2) whether the extent of the decline differed among clutches according to laying date. Our results show that yolk lutein decreased along the laying sequence at the clutch level. More interestingly, they also show that the decrease in yolk lutein levels along the laying sequence was more pronounced in late clutches than in early clutches, although lutein levels in adult plasma did not vary according to laying date. Overall, these results support the hypothesis that the Yellow-legged Gull employs the brood reduction strategy and that said strategy is more drastically applied in late clutches than in early clutches. The relevance of female age and seasonal variation in chick-rearing conditions for these findings is discussed.

Neuroactive steroid levels in plasma and cerebrospinal fluid of male multiple sclerosis patients

Neuroactive steroid family includes molecules synthesized in peripheral glands (i.e., hormonal steroids) and directly in the nervous system (i.e., neurosteroids) which are key regulators of the nervous function. As already reported in clinical and experimental studies, neurodegenerative diseases affect the levels of neuroactive steroids. However, a careful analysis comparing the levels of these molecules in cerebrospinal fluid (CSF) and in plasma of multiple sclerosis (MS) patients is still missing. To this aim, the levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in CSF and plasma of male adults affected by Relapsing-Remitting MS and compared with those collected in control patients. An increase in pregnenolone and isopregnanolone levels associated with a decrease in progesterone metabolites, dihydroprogesterone, and tetrahydroprogesterone was observed in CSF of MS patients. Moreover, an increase of 5α-androstane-3α,17β-diol and of 17β-estradiol levels associated with a decrease of dihydrotestosterone also occurred. In plasma, an increase in pregnenolone, progesterone, and dihydrotestosterone and a decrease in dihydroprogesterone and tetrahydroprogesterone levels were reported. This study shows for the first time that the levels of several neuroactive steroids, and particularly those of progesterone and testosterone metabolites, are deeply affected in CSF of relapsing-remitting MS male patients. We here demonstrated that, the cerebrospinal fluid and plasma levels of several neuroactive steroids are modified in relapsing remitting multiple sclerosis male patients. Interestingly, we reported for the first time that, the levels of progesterone and testosterone metabolites are deeply affected in cerebrospinal fluid. These findings may have an important relevance in therapeutic and/or diagnostic field of multiple sclerosis.

Early adolescent stress alters behavior and the HPA axis response in male and female adult rats: the relevance of the nature and duration of the stressor

Adolescence is a period of transition from childhood to adulthood that involves the maturation of social and cognitive behavior. The activation of the stress system during this phase can lead to long-lasting adverse effects. We aimed to verify whether the nature and duration of stressors applied in adolescent female and male rats would alter their exploratory behavior and stress responses as adults. Wistar rats on day P26 were divided into groups that were subjected to 1 (acute) or 7 (chronic) insulin or lipopolysaccharide (LPS) injections or restraint stress for 1h. At P60, the rats were subjected to the elevated plus-maze, and at P61, they were subjected to 30min of restraint stress after which plasma samples and brains were collected. LPS acute injection promoted anxiolytic effects in male adults. Acute LPS treatment and acute or chronic restraint induced anxiolytic behavior in female adults. The administration of adolescent chronic stimuli to males decreased the adult plasma corticosterone (CORT) and progesterone levels after restraint. Adolescent acute restraint or LPS injection decreased the CORT response in female adults. The adult neuronal activation of the corticotrophin-releasing hormone and vasopressin on the paraventricular nucleus did not vary according to the type of adolescent stress or sex. Our results indicate that both adult behavior and the glucocorticoid stress response are affected differently in males versus females by adolescent stress. The duration of stressors had a greater effect on the CORT and progesterone response in males, whereas the nature of the stressor had a greater effect on exploratory behavior in females.

Inadequate passive immune transfer in puppies: definition, risk factors and prevention in a large multi-breed kennel

The prevalence of neonatal mortality is high in the canine species and far from well-studied. In most domestic neonates, an appropriate colostrum intake is a key element of the control of neonatal mortality. The aim of this study was to evaluate the impact of passive immune transfer on puppy mortality, assessed through serum immunoglobulin G (IgG) concentration at 2 days of age. Factors impacting passive immune transfer and the value of an oral immunoglobulin supplementation to prevent it were also analyzed. A total of 149 puppies from 34 litters (12 breeds) within one breeding kennel were included. Blood samples were collected at 2 days of age and colostrum was collected from their dams 1 day after whelping to assay IgG concentration. Puppies were weighed at birth and at 2 days of age for calculation of growth rate. Mortality was recorded until 3 weeks of age. Seventy randomly assigned puppies were orally supplemented with hyper-immunized adult plasma twice within the first 8h of life. IgG concentration at 2 days of age was significantly correlated with weight gain during the first 2 days of life. The multivariable model with litter as a random effect demonstrated that neonatal mortality was not influenced by breed size, sex, supplementation, litter size, nor colostrum IgG concentration, but by puppy IgG concentration at 2 days of age. According to the ROC curve, the minimal IgG concentration at and below which puppies were at higher risk of death was determined at 230mg/dl. Puppy IgG concentration was significantly associated with growth rate, but not with breed size, sex, supplementation, litter size or colostrum IgG concentration in a multivariable model with litter as a random effect. This study demonstrates that neonatal mortality in puppies is related to the quality of passive immune transfer. The oral supplementation with hyper-immunized canine plasma neither decreased risk of mortality, nor improved serum IgG concentration at 2 days of age in puppies. Attention must thus be paid to early colostrum intake to control the neonatal mortality in puppies.

The in-vitro anticoagulant effect of rivaroxaban in neonates

The use of anticoagulants in neonates is increasing because of the medical advances improving the long-term survival of very sick infants who are at risk of venous thromboembolism (VTE). Current anticoagulation therapy in neonates is less than ideal, because of the physiological differences compared to children and adults regarding the pathophysiology of thrombosis and pharmacology of the drug. Limitations associated with conventional anticoagulants have prompted the development of novel drugs that specifically target the key proteins in the coagulation system. Rivaroxaban is the first oral, direct Factor Xa inhibitor available for the prevention of VTE in adults. Its predictable pharmacokinetic profile, high oral bioavailability and once-daily dosing make rivaroxaban an optimal anticoagulant that warrants investigation in neonates. This study was designed to determine whether there are age-related differences in the pharmacodynamic effects of rivaroxaban in vitro amongst neonates. Neonatal and adult plasma pools were created and spiked with increasing concentrations of rivaroxaban (0-500 ng/ml). Commercially available prothrombin time (PT), activated partial thromboplastin time (aPTT) and anti-Factor Xa assays as well as a sub-sampling thrombin generation assay were used to measure the rivaroxaban effect. A dose-dependent response was observed for PT, aPTT and lag time in both the age groups. Rivaroxaban caused a significant increase in the clotting time for PT and aPTT as well as an increase in lag time (as measured by thrombin generation) in neonates when compared with adults. In-vivo studies are required to confirm the consistency of dose-response in neonates.

Physiologically based pharmacokinetic modelling of methotrexate and 6-mercaptopurine in adults and children. Part 2: 6-mercaptopurine and its interaction with methotrexate

6-mercaptopurine (6-MP) is a purine antimetabolite and prodrug that undergoes extensive intracellular metabolism to produce thionucleotides, active metabolites which have cytotoxic and immunosuppressive properties. Combination therapies involving 6-MP and methotrexate have shown remarkable results in the cure of childhood acute lymphoblastic leukaemia(ALL) in the last 30years. 6-MP undergoes very extensive intestinal and hepatic metabolism following oral dosing due to the activity of xanthine oxidase leading to very low and highly variable bioavailability and methotrexate has been demonstrated as an inhibitor of xanthine oxidase. Despite the success recorded in the use of 6-MP in ALL, there is still lack of effect and life threatening toxicity in some patients due to variability in the pharmacokinetics of 6-MP. Also, dose adjustment during treatment is still based on toxicity. The aim of the current work was to develop a mechanistic model that can be used to simulate trial outcomes and help to improve dose individualisation and dosage regimen optimisation. A physiological based pharmacokinetic model was proposed for 6-MP, this model has compartments for stomach, gut lumen, enterocyte, gut tissue, spleen, liver vascular, liver tissue, kidney vascular, kidney tissue, skin, bone marrow, thymus, muscle, rest of body and red blood cells. The model was based on the assumption of the same elimination pathways in adults and children. Parameters of the model include physiological parameters and drug-specific parameter which were obtained from the literature or estimated using plasma and red blood cell concentration data. Age-dependent changes in parameters were implemented for scaling and variability was also introduced on the parameters for prediction. Inhibition of 6-MP first-pass effect by methotrexate was implemented to predict observed clinical interaction between the two drugs. The model was developed successfully and plasma and red blood cell concentrations were adequately predicted both in terms of mean prediction and variability. The predicted interaction between 6-MP and methotrexate was slightly lower than the reported clinical interaction between the two drugs. The model can be used to predict plasma and tissue concentration in adults and children following oral and intravenous dosing and may ultimately help to improve treatment outcome in childhood ALL patients.

Plasma and Serum Lipidomics of Healthy White Adults Shows Characteristic Profiles by Subjects’ Gender and Age

Blood is a commonly used biofluid for biomarker discovery. Although blood lipid metabolites are considered to be potential biomarker candidates, their fundamental properties are not well characterized. We aimed to (1) investigate the matrix type (serum vs. plasma) that may be preferable for lipid biomarker exploration, (2) elucidate age- and gender-associated differences in lipid metabolite levels, and (3) examine the stability of lipid metabolites in matrix samples subjected to repeated freeze-thaw cycles. Using liquid chromatography-mass spectrometry, we performed lipidomic analyses for fasting plasma and serum samples for four groups (15 subjects/group) of young and elderly (25–34 and 55–64 years old, respectively) males and females and for an additional aliquot of samples from young males, which were subjected to repeated freeze-thaw cycles. Lysophosphatidylcholine and diacylglycerol levels were higher in serum than in plasma samples, suggesting that the clotting process influences serum lipid metabolite levels. Gender-associated differences highlighted that the levels of many sphingomyelin species were significantly higher in females than in males, irrespective of age and matrix (plasma and serum). Age-associated differences were more prominent in females than in males, and in both matrices, levels of many triacylglycerols were significantly higher in elderly females than in young females. Plasma and serum levels of most lipid metabolites were reduced by freeze-thawing. Our results indicate that plasma is an optimal matrix for exploring lipid biomarkers because it represents the original properties of an individual’s blood sample. In addition, the levels of some blood lipid species of healthy adults showed gender- and age-associated differences; thus, this should be considered during biomarker exploration and its application in diagnostics. Our fundamental findings on sample selection and handling procedures for measuring blood lipid metabolites is important for ensuring the quality of biomarkers identified and its qualification process.

Effect of age and ration on diet-tissue isotopic (Δ13C, Δ15N ) discrimination in striped skunks (Mephitis mephitis)

An important prerequisite for the effective use of stable isotopes in animal ecology is the accurate assessment of isotopic discrimination factors linking animals to their diets for a multitude of tissue types. Surprisingly, these values are poorly known in general and especially for mammalian carnivores and omnivores in particular. Also largely unknown are the factors that influence diet-tissue isotopic discrimination such as nutritional quality and age. We raised adult and juvenile striped skunks (Mephitis mephitis) in captivity on a constant omnivore diet (Mazuri Omnivore A 5635). Adults (n=6) and juveniles (n=3) were kept for 7 months and young (n=7) to the age of 50 days. We then examined individuals for stable carbon (δ13C) and nitrogen (δ15N) isotope values of hair, nails, lipid, liver, muscle, bone collagen and the plasma, and cellular fractions of blood. Discrimination values differed among age groups and were significantly higher for young compared with their mothers, likely due to the effects of weaning. Δ15N isotopic discrimination factors ranged from 3.14 (nails) to 5.6‰ (plasma) in adults and 4.3 (nails) to 5.8‰ (liver) for young. For Δ13C, values ranged from−3.3 (fat) to 3.0‰ (collagen) in adults and from−3.3 (fat) to 2.0‰ (collagen) in young. Our data provide an important tool for predicting diets and source of feeding for medium-sized mammalian omnivorous adults integrated over short (e.g. liver, plasma) through long (e.g. collagen) periods and underline the potential effects of age on isotopic values in omnivore diets.

Top Advances in Functional Genomics and Translational Biology for 2013

The year 2013 has witnessed exciting advances in cardiovascular research exemplified by discovery leveraging genomics, proteomics and metabolomics platforms, genome editing, translation in individualized disease modeling, and application of regenerative therapies. New knowledge generated through integrative analysis has informed the understanding of molecular mechanisms that can now be validated and exploited to expedite management of biological processes. The Functional Genomics and Translational Biology Council from the American Heart Association (www.my.americanheart.org/fgtbcouncil) has the mission of advancing new discoveries in the fields of genetics, omics technologies, and translational biology and facilitate their application in cardiovascular health and disease. By creating a multidisciplinary collaborative environment, this Council integrates scientific knowledge from molecules to populations to contribute to the global American Heart Association goal of building healthier lives, free of cardiovascular diseases and stroke. With input from the Early Career Committee of the Council on Functional Genomics and Translational Biology, we considered many outstanding articles and selected 10 advances published during 2013. In this Special Report, we summarize some relevant articles related to each of those advances and highlight their significance in the cardiovascular field. Cardiac hypertrophy is an increasingly prevalent condition because of population aging with limited therapeutic options to prevent its progression to heart failure. Loffredo et al1 hypothesized that loss of systemic factors may be responsible for the stiffening and enlargement of the ventricles and thereby a therapeutic target for the treatment of systolic heart failure. Using a model of parabiosis (surgically linking the systemic circulation of 2 animals), they demonstrated that exposure to a young circulatory environment can reverse age-induced cardiac hypertrophy in old mice. Proteomic analysis of old and young adult plasma enabled identification of the growth differentiation factor 11, a member of the transforming growth factor-β superfamily, as a factor with decreasing blood levels in aging mice. …

Plasma and Red Cell Reference Intervals of 5-Methyltetrahydrofolate of Healthy Adults in Whom Biochemical Functional Deficiencies of Folate and Vitamin B12Had Been Excluded

5-Methyltetrahydrofolate (5-MTHF) is the predominant form of folate and a strong determinant of homocysteine concentrations. There is evidence that suboptimal 5-MTHF availability is a risk factor for cardiovascular disease independent of homocysteine. The analysis of folates remains challenging and is almost exclusively limited to the reporting of “total” folate rather than individual molecular forms. The purpose of this study was to establish the reference intervals of 5-MTHF in plasma and red cells of healthy adults who had been prescreened to exclude biochemical evidence of functional deficiency of folate and/or vitamin B12. Functional folate and vitamin B12 status was assessed by respective plasma measurements of homocysteine and methylmalonic acid in 144 healthy volunteers, aged 19–64 years. After the exclusion of 10 individuals, values for 134 subjects were used to establish the upper reference limits for homocysteine (13 μmol/L females and 15 μmol/L males) and methylmalonic acid (430 nmol/L). Subjects with values below these cutoffs were designated as folate and vitamin B12 replete and their plasma and red cell 5-MTHF reference intervals determined, N = 126: 6.6–39.9 nmol/L and 223–1041 nmol/L, respectively. The application of these intervals will assist in the evaluation of folate status and facilitate studies to evaluate the relationship of 5-MTHF to disease.

Inhibition of tumour spheroid-induced prometastatic intravasation gates in the lymph endothelial cell barrier by carbamazepine: drug testing in a 3D model

Metastatic breast cancer is linked to an undesired prognosis. One early and crucial metastatic step is the interaction of cancer emboli with adjacent stroma or endothelial cells, and understanding the mechanisms of this interaction provides the basis to define new targets as well as drugs for therapy and disease management. A three-dimensional (3D) co-culture model allowing the examination of lymphogenic dissemination of breast cancer cells was recently developed which facilitates not only the study of metastatic processes but also the testing of therapeutic concepts. This 3D setting consists of MCF-7 breast cancer cell spheroids (representing a ductal and hormone-dependent subtype) and of hTERT-immortalised lymph endothelial cell (LEC; derived from foreskin) monolayers. Tumour spheroids repel the continuous LEC layer, thereby generating "circular chemorepellent-induced defects" (CCIDs) that are reminiscent to the entry gates through which tumour emboli intravasate lymphatics. We found that the ion channel blocker carbamazepine (which is clinically used to treat epilepsy, schizophrenia and other neurological disorders) inhibited CCID formation significantly. This effect correlated with the inhibition of the activities of NF-κB, which contributes to cell motility, and with the inactivation of the mobility proteins MLC2, MYPT1 and FAK which are necessary for LEC migration. NF-κB activity and cell movement are prerequisites of CCID formation. On the other hand, the expression of the motility protein paxillin and of the NF-κB-dependent adhesion mediator ICAM-1 was unchanged. Also the activity of ALOX12 was unaffected. ALOX12 is the main enzyme synthesising 12(S)-HETE, which then triggers CCID formation. The relevance of the inhibition of CYP1A1, which is also involved in the generation of mid-chain HETEs such as 12(S)-HETE, by carbamazepine remains to be established, because the constitutive level of 12(S)-HETE did not change upon carbamazepine treatment. Nevertheless, the effect of carbamazepine on the inhibition of CCID formation as an early step of breast cancer metastasis was significant and substantial (~30 %) and achieved at concentrations that are found in the plasma of carbamazepine-treated adults (40-60 μM). The fact that carbamazepine is a drug approved by the US Food and Drug Administration facilitates a "from-bench-to-bedside" perspective. Therefore, the here presented data should undergo scrutiny in vivo.

Development of a Standard Reference Material for metabolomics research.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Apheresis activity in Spain: A survey of the Spanish Apheresis Group

The Spanish Apheresis Group is a scientific association of physicians and nurses representing most of the medical centers in the country that are involved in apheresis. The group developed a survey in order to get information about the types and number of apheresis procedures performed in Spain. We received responses from 66 centers and we were able to collect data from at least one center of each autonomous region. There were 7 centers (11%) that did not perform any kind of apheresis procedures, 26 (39%) centers performed therapeutic apheresis procedures only, 18 (27%) centers performed apheresis donations only, and 15 (23%) centers performed both types of apheresis procedures. Regarding therapeutic apheresis in adult patients, plasma exchange (34%) and stem cell collections (30%) were the two therapeutic procedures most frequently reported, followed by erythrocytapheresis (13%) and extracorporeal photochemotherapy (11%). Regarding apheresis donation, our survey showed that the most frequent was multicomponent donation (45%) followed by plasmapheresis (28%) and single plateletapheresis (21%). When analyzing the current instrumentation for performing apheresis procedures, centers used the Spectra, Optia, and Trima devices (TerumoBCT) as the most frequent ones, followed by the MCS+(Haemonetics), Amicus (Fenwal), and Fresenius devices. In conclusion, we report here the first nationwide survey performed in Spain in order to get information about apheresis activities in our country. The survey is representative of Spain because we were able to collect data from at least one center from each of the different 17 autonomous regions, and we found a wide variety of therapeutic and donation procedures, as well as instrumentation used.

The Effective Concentration of Tranexamic Acid for Inhibition of Fibrinolysis in Neonatal Plasma In Vitro

Neonates are at high risk for bleeding complications after cardiovascular surgery. Activation of intravascular fibrinolysis is one of the principal effects of cardiopulmonary bypass that causes poor postoperative hemostasis. Antifibrinolytic medications such as tranexamic acid are often used as prophylaxis against fibrinolysis, but concentration/effect data to guide dosing are sparse for adults and have not been published for neonates. Higher concentrations of tranexamic acid than those necessary for inhibition of fibrinolysis may have adverse effects. Therefore, we investigated the concentration of tranexamic acid necessary to inhibit activated fibrinolysis in neonatal plasma. We conducted an in vitro study using neonatal plasma derived from the placenta/cord units from 20 term, elective cesarean deliveries. Graded concentrations of tranexamic acid were added to aliquots of the pooled plasma before maximally activating fibrinolysis with high-dose tissue-type plasminogen activator. Thromboelastography was then performed with the primary outcome variable being lysis at 30 minutes. These procedures were repeated on pooled adult normal plasma and dilutions of neonatal plasma. The minimum concentrations of tranexamic acid to completely prevent fibrinolysis were 6.54 μg/mL (95% confidence interval, 5.19-7.91) for neonatal plasma and 17.5 μg/mL (95% confidence interval, 14.59-20.41) for adult plasma. Neonatal plasma requires a significantly lower concentration than adult plasma (P < 0.0001, 2-sided Wald test). Our data establish the minimal effective concentration of tranexamic acid necessary to completely prevent fibrinolysis in neonatal plasma in vitro. These data may be useful in designing a dosing scheme for tranexamic acid appropriate for neonates.

Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase, and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.

Cord blood neutrophils display a galectin-3 responsive phenotype accentuated by vaginal delivery

BackgroundTerm neonates are at increased risk of infections due to undeveloped immune mechanisms, and proper neutrophil function is important for perinatal immune defence. Galectin-3, an endogenous β-galactoside-binding lectin, is emerging as an inflammatory mediator and we have previously shown that primed/activated, but not resting, adult neutrophils respond to this lectin by production of reactive oxygen species (ROS). We investigated if galectin-3 is of importance in perinatal immune defence, focusing on plasma levels and neutrophil responsiveness.MethodsNeutrophils were isolated from peripheral blood of healthy adults and cord blood (CB) after elective Caesarean section (CSCB) and vaginal delivery (VDCB). ROS production was measured by chemiluminescence, L-selectin expression by flow cytometry, and interleukin-8 (IL-8) and galectin-3 concentrations by ELISA. Statistical evaluations were performed using the Mann–Whitney test.ResultsIn response to galectin-3, CSCB neutrophils showed a small but clear ROS production not evident in adult cells, signifying that neonatal neutrophils exist in a primed state. IL-8 production was elevated in CSCB cells while L-selectin exposure was equal to adult cells. Comparing CSCB to VDCB neutrophils, the latter showed an extensive galectin-3 responsiveness, indicating that the degree of priming is dependent on mode of delivery. VDCB neutrophils were increasingly prone to shed L-selectin, while the amount of IL-8 was similar to CSCB cells. The endogenous galectin-3 levels were higher in neonatal as compared to adult plasma, unaffected by mode of delivery.ConclusionsNeutrophils enter a pre-primed state already in the fetus. Upon exposure to the inflammatory stimuli that are associated with labor, the neutrophils develop a reactive phenotype with extensive priming features.