DPG Levels Research Articles

Presence of 1, 3-diphenylguanidine and its derivatives in human urine and their human exposure

Recent studies have demonstrated the widespread presence of 1,3-diphenylguanidine (DPG) and its derivatives in environmental matrices. While, the amount of human exposure to these rubber additives remains unclear. In this study, we collected human urine samples from healthy general adults (n = 221) living in Quzhou, China, and analyzed these samples for DPG and its five derivatives. DPG, 1,6-bis(cyano-guanidino)hexane (HCG), 1,3-di-o-tolylguanidine (DTG) and exhibited detection frequencies exceeding 50% in collected human urine. Presence of HCG, 1-(o-tolyl)biguanide (detection frequency 17%), and 1-(4-cyanophenyl)guanidine (6.0%) in human urine was also demonstrated for the first time. The highest mean human urinary concentration was found for DPG (0.89 ng/mL, < LOD−4.7 ng/mL), followed by DTG (0.57 ng/mL, < LOD−3.1 ng/mL) and HCG (0.34 ng/mL, < LOD−1.8 ng/mL). Male participants had consistently higher average human urinary levels of DPG, DTG, and HCG than female subjects, but none of these differences were significant (p > 0.10). DPG and DTQ consistently showed a decline in the human urinary concentrations as age of the participant increased. DPG (mean 170 ng/kg bw/day, median 137 ng/kg bw/day) had the highest human daily exposure amount, followed by DTG (106 ng/kg bw/day, 91 ng/kg bw/day) and HCG (58 ng/kg bw/day, 38 ng/kg bw/day). The study enhances our understanding of human exposure to these rubber additives, which is crucial for assessing their potential health risks.

Sphingosine-1-phosphate Decreases Erythrocyte Dysfunction Induced by β-Amyloid.

Amyloid beta peptides (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Soluble Aβ oligomers, rather than monomer or insoluble amyloid fibrils, show red blood cell (RBC) membrane-binding capacity and trigger several morphological and functional alterations in RBCs that can result in impaired oxygen transport and delivery. Since bioactive lipids have been recently proposed as potent protective agents against Aβ toxicity, we investigated the role of sphingosine-1-phosphate (S1P) in signaling pathways involved in the mechanism underlying ATP release in Ab-treated RBCs. In RBCs following different treatments, the ATP, 2,3 DPG and cAMP levels and caspase 3 activity were determined by spectrophotometric and immunoassay. S1P rescued the inhibition of ATP release from RBCs triggered by Ab, through a mechanism involving caspase-3 and restoring 2,3 DPG and cAMP levels within the cell. These findings reveal the molecular basis of S1P protection against Aβ in RBCs and suggest new therapeutic avenues in AD.

Biochemical and Metabolomic Analysis of Glycolytic Activity in Red Blood Cells from Low-Risk Myelodysplastic Syndromes (LR-MDS) Patients and in-Vitro Effect of the Pyruvate Kinase Activator AG-946

Background: Anemia is the most common cytopenia occurring in 80%-85% of patients (pts) with LR-MDS and therapeutic strategies remain limited. Beyond defective maturation, preliminary data has shown decreased glycolytic activity in MDS-RBCs. Glycolytic pathway is the prominent metabolic cascade in RBCs and is currently targeted with PK activators in clinical trials of congenital anaemias which are also marked by ineffective erythropoiesis like MDS. AG946 is an investigational, potent activator of PK with potential to enhance RBC functionality and survival by increasing glycolysis. Aim: Evaluate enzymatic activity of the glycolytic pathway in pts with anemia due to LR-MDS and in-vitro effect of pyruvate kinase (PK) activator (AG946) in RBCs cultures of LR-MDS pts. Methods In part A, 66 anemic pts (i.e., Hb&amp;lt;11 g/dL) diagnosed with MDS according to WHO 2022 were enrolled in the study and sampled for peripheral blood &amp;gt;4 weeks after the last RBC unit to reduce blood donor contamination. The determination of activities of RBC enzymes hexokinase (HK) and PK (the initial and terminal steps of glycolytic pathway), and of ATP levels was performed by standard spectrophotometric method according to Beutler et al, 1984. 2,3DPG levels were tested by (2,3-BPG) ELISA kit (ElisaGenie, IE). In part B, purified RBC or whole blood of 10 LRMDS pts and 5 healthy controls (HC) were incubated for 6 and 24 h at 37°C in presence or absence of AG946 in phosphate-buffered saline containing 1% glucose, 170 mg/L adenine, and 5.25 g/L mannitol (AGAM, pH 7.40). RBC were incubated with 1, 5 and 50 uM of AG946 for up to 24 h, at 37°C. After 6 and 24 h, enzymatic activities (HK, PK) and ATP levels were measured. Additionally, metabolomic analysis was performed by LC-MS on extracts from blood samples for each condition. Results Part A: Pts were mainly elderly males (median age 80yr, range 58-92), and mostly belonged to IPSS-R low (78%) and very low/low groups (85%). PK activity (median 13.7 IU/gHb, 7.5-35.9) was reduced in 35% of cases and HK activity (median 2.3 IU/gHb, 0.8-8.1) was increased in 91%, resulting in an abnormally reduced PK/HK ratio in majority of pts (95%). Notably, ATP levels (median 3.4 umol/gHb, 1.3-6.5) were also reduced in 33/56 tested patients (59%), whilst 2,3DPG levels were close to the upper limit of the normal range (median 389 nmol/gHb, 318-639). Conclusions: Our data demonstrates decreased glycolytic activity in a large cohort of anemic pts with LR-MDS versus HC, with reduced PK activity in 1/3 of cases, decreased PK/HK ratio in nearly all subjects, and reduced ATP in 2/3 of pts. Furthermore, in vitro incubation with AG946 led to an increase in PK activity and PK/HK ratio and stable ATP levels in RBCs of LR-MDS pts across all WHO classifications. Metabolomic analysis confirmed an altered RBC-metabolic status of MDS versus HC at baseline with possible modulation of glycolysis by AG946, further supporting a potential therapeutic role of PK Activation with AG946 in LR-MDS.

Quantitation of guanidine derivatives as representative persistent and mobile organic compounds in water: method development

Persistent and mobile organic compounds (PMOCs) are highly soluble in water, thereby posing a threat to water resource quality. Currently, there are no methods that can accurately quantify guanidine derivative PMOCs, other than 1,3-diphenylguanidine (DPG) and cyanoguanidine (CG), in aqueous media. In this study, we developed a quantitation method that combines solid-phase extraction and liquid chromatography (LC)-tandem mass spectrometry to detect seven guanidine derivatives in aquatic environments and applied it to environmental water samples. Five LC columns were examined, and among them, a hydrophilic interaction liquid chromatography column was chosen owing to its suitable instrument detection limit and retention factor. Method precision was assessed using seven replicate analyses of river water. The corresponding analyte recoveries ranged from 73 to 137% (coefficient of variation = 2.1–5.8%). DPG and CG were detected in ultrapure water samples at levels up to 0.69 and 150 ng L−1, respectively; DPG and CG levels up to 44 and 2600 ng L−1, respectively, were detected in lake water, river water, sewage effluent, and tap water sampled in Western Japan. This is the first reported detection of DPG in the surface water of Japan, revealing that DPG and CG are ubiquitous compounds in aquatic environments. Moreover, this is the first study to detect 1-(o-tolyl)biguanide and N,N′′′-1,6-hexanediylbis(N′-cyanoguanidine) in water. This study provides a foundation for further research on the distribution, fate, and emission source of these pollutants, which is critical to maintain high water quality and to determine regulatory limits for these pollutants.Graphical

Evaluation of Red Blood Cell Metabolism in Patients with Low-Risk Myelodysplastic Syndrome (LR-MDS): A Proof-of-Concept Study

Low risk myelodysplastic syndromes (MDS) show high clinical heterogeneity ranging from mild anemia with nearly normal performance status to life-threatening cytopenias, frequent hospitalization, and disability. Anemia occurs in 80%-85% of patients and therapeutic strategies are still very limited mainly consisting of of erythropoiesis stimulating agents and red blood cell (RBC) transfusion support. Starting from the working hypothesis that the defective maturation of hematopoietic precursors may dampen the "functionality" of MDS-RBCs, we aimed at evaluating RBC metabolic pathway in anemic patients with LR-MDS. We focused on the glycolytic pathway, which is the prominent metabolic cascade in RBCs and is currently targeted with allosteric stimulators of pyruvate kinase (PK) enzyme in clinical trials of PK deficiency, thalassemia, and sickle cell diseases, which are also marked by ineffective erythropoiesis like MDS. Sixty-six consecutive anemic patients (i.e. Hb<11 g/dL) diagnosed with MDS according to WHO 2016 classification that met IPSS-R criteria for very low, low, or intermediate-risk MDS were enrolled in the study. All patients were sampled for peripheral blood at least 4 weeks after the last RBC unit to reduce possible blood donor contamination. The determination of activities of the glycolytic RBC enzymes pyruvate kinase (PK) and hexokinase (HK) (the terminal and initial steps of the glycolytic pathway), and of ATP levels was performed by standard spectrophotometric method according to Beutler et al, 1984. 2,3 DPG determination was tested by AssayGenie Kit in a subgroup of patients. Ektacytometric analysis by LoRRca MaxSis (Laser-Assisted Optical Rotational Cell Analyzer, Mechatronics, NL) was also performed in a subgroup of patients. As shown in table 1, patients were mainly elderly males, and mostly belonged to IPSS low (78%) and IPSS-R very low/low groups (85%). Median levels of PK activity were 13.7 IU/gHb, with a wide range (7.5-35.9) and were reduced in 23 cases (35%; normal reference range 11.9-16.7 IU/gHb); median values of HK activity were 2.3 (0.8-8.1), increased in 60 patients (91%; normal reference range 0.78-1.32 IU/gHb). This resulted in an abnormally reduced PK/HK ratio (median 5.45, range 2-15) in 63 subjects (95%; (normal reference range calculated on 167 normal subjects: 11.55 - 17.65). To asses if impaired glycolysis may result in altered RBC deformability or in lack of energy, ATP levels, 2,3 DPG, and ektacytometric curve were evaluated. Median ATP levels were 3.4 mcM/gHb (1.3-6.5), reduced in 33/56 tested patients (59%; normal reference range 3.6-4.9 mcM/gHb), of whom 32 had simultaneous reduction of PK/HK activity ratio (figure 1), and 11 decreased PK activity. Concerning 2,3 DPG levels preliminarily analysed in a subgroup of patients, they were close to the upper limit of the normal range (median, 389 nM/gHb, 318-639). Osmoscan, performed in 10 patients did not show altered RBC deformability. Preliminary data show decreased glycolytic activity in RBCs from patients with LR-MDS highlighted by reduced PK activity in more than 1/3 of cases, and increased HK activity in almost all subjects. This results in reduced RBC ATP content in up to 2/3 of patients (possibly due to reduced production or to increased consumption) indicating an overall lack of energy in MDS-RBCs. The latter may be an interesting target for allosteric PK stimulators and warrants further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Apolipoprotein A1 is associated with pulmonary vascular resistance and adverse clinical outcomes in patients with pulmonary hypertension secondary to heart failure.

Pulmonary hypertension secondary to heart failure (HF‐PH) combined with pulmonary vascular remodeling has a high mortality rate. Apolipoprotein A1 (ApoA1) has been shown to adversely affect outcomes in patients with HF. A prospective follow‐up study was performed on 239 consecutive patients with HF‐PH who underwent right heart catheterization. Proteomics technology was used to analyze different proteins in plasma between post‐ and precapillary pulmonary hypertension (CpcPH) and isolated postcapillary pulmonary hypertension (IpcPH) filtered by propensity score matching. Ultimately, 175 patients were enrolled and followed for an average of 4.4 years. Lipoprotein components in plasma were measured, and the following clinical events were tracked. Proteomics data showed that lipid metabolism and inflammation were different between CpcPH and IpcPH. ApoA1 levels in HF‐PH patients with CpcPH were lower than those in HF‐PH patients with IpcPH. The patients with lower ApoA1 levels (≤1.025 g/L) were in a higher New York Heart Association class and had high levels of NT‐proBNP, mean pulmonary artery pressure, PVR, and diastolic pressure gradient. Besides, HF‐PH patients with lower ApoA1 levels had a 2.836‐fold higher relative risk of comorbid CpcPH compared with patients with higher ApoA1 levels. Moreover, patients with lower ApoA1 levels had a lower survival rate after adjusting for CpcPH. In conclusion, ApoA1 levels were negatively correlated with PVR levels. Lower ApoA1 levels were an independent risk factor for pulmonary vascular remodeling in HF‐PH patients. The survival of HF‐PH patients with lower ApoA1 levels was reduced.

Effect of Hematocrit Injury on the Survival Rate of Advanced Malignant Tumors and Its Clinical Significance.

Objective To investigate the effect of logistic multivariate analysis on the survival rate of advanced malignant tumors and to evaluate the effect of erythrocyte storage injury on the survival rate of advanced malignant tumors and its clinical significance. Methods A retrospective analysis was performed on 120 advanced cancer patients who received blood transfusion in Shaanxi Cancer Hospital from March 2018 to June 2019, and the risk factors for death were analyzed. A total of 72 advanced cancer patients admitted to hospital from March 2019 to June 2021 were included in the study. The patients with red blood cell transfusion storage time ≤ 14 d were the study group (n = 36), and the patients with red blood cell transfusion storage time > 14 d were the control group (n = 36). Compare the total efficiency of blood transfusion. The levels of Hb, erythrocyte count, hematocrit (HCT), blood oxygen saturation (SPO2), creatinine (Cr), erythrocyte deformability index, whole blood, erythrocyte, and hemoglobin before and after blood transfusion were compared, and the adverse reactions of blood transfusion were recorded. Results Dyspnea and delirium were significantly associated with patient survival time (P < 0.05). Red blood cell storage time ≤ 14 days, Lym% < 12%, lactate dehydrogenase (LDH) > 500 U/L, and ALB < 30 g/L were significantly correlated with survival time. Karnofsky performance status (KPS) < 30, delirium, LDH > 500 U/L, and albumin (ALB) < 30 g/L were independent influencing factors of survival (P < 0.05). The overall effective rate of the research group was higher (P < 0.05). The incidence of adverse reactions in the study group was lower (P < 0.05). The levels of Hb, red blood cell count, and HCT in the study group were higher (P < 0.05). Compared with the control group, the SPO2 level and the red blood cell deformability index were higher in the study group (P < 0.05). After blood transfusion, the level of (diphosphoglycerate) DPG in the study group was higher than that in the control group (P < 0.05). The length of hospital stay in the study group was significantly shortened (P < 0.05). The nosocomial infection rate and case fatality rate in the study group were significantly reduced (P < 0.05). Conclusion Red blood cell storage time ≤ 14 d, LYM% < 12%, LDH > 500 U/L, and ALB < 30 g/L are all significantly correlated with survival time. KPS < 30, delirium, LDH > 500 U/L, and ALB < 30 g/L were independent factors for survival (P < 0.05). Transfusion of red blood cells stored for ≤14 days in patients with advanced malignant tumors can significantly increase the effective infusion rate, improve anemia status, shorten hospital stay, and reduce mortality and risk of nosocomial infection and is worthy of clinical promotion.

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Tibetan Enriched PKLR Variant Is Beneficial to High Altitude Adaption By Improving Oxygen Delivery

Tibetans have been living at altitudes over 3500 m for ~20,000 years and have developed unique beneficial evolutionary genetic adaptions (PMID:28448578). Our previous study identified selected genetic haplotypes in two genes, EPAS1 (encoding hypoxia-inducible factor 2-alpha [HIF2-a], a transcription factor that mediates the hypoxic response), and EGLN1 (encoding prolyl hydroxylase 2 [PHD2], a principal negative regulator of HIF stability (PMID:25129147). The presence of these two haplotypes correlates with lower hemoglobin levels in Tibetans compared to other highlanders. However, the entire diverse complex of molecular mechanisms of high altitude adaptation is still largely unknown and our study showed that neither EPAS1 nor EGLN1 variants fully explain the mechanism of protection from polycythemia in Tibetans in high altitude (PMID:28233034).We found an enriched haplotype in the PKLR gene (encoding pyruvate kinase [PK] expressed only in liver and red blood cells). The PK enzyme is in the terminal portion of the glycolytic pathway, and its decreased activity leads to accumulation of proximal glycolytic intermediates, including 2,3-diphosphoglycerate (DPG) which shifts the hemoglobin dissociation curve to right (high p50) and increases oxygen release to tissues from a unit of hemoglobin. We hypothesized that Tibetan enriched PKLR variants might improve oxygen delivery to tissues and help explain the protection from polycythemia at high altitude.Genomic analyses revealed that this PKLR haplotype is enriched in Tibetans but is not unique to Tibetans. It has the highest frequency in Tibetans (89%), with a lower prevalence in Chinese and Mongolians (~77%), Kyrgyz (~60 %), Aymara (~44 %), and Colombians (~30 %) and a much lower frequency in Caucasians (11%), perhaps explaining the heterogeneity of responses to hypoxia within and among these populations. Our study of reticulocyte RNA showed that transcript levels of PKLR progressively decrease with increasing altitude in controls and even more in Tibetans with the Tibetan evolutionary selected PKLR haplotype. Tibetans with the PKLR haplotype (heterozygotes and homozygotes) have lower PKLR transcript levels than wild type Tibetans. Because of the paucity of wildtype PKLR haplotype in Tibetans and the challenges of acquiring Tibetan samples in high altitude in China, we collected samples from 125 m (Cheboksary, Chuvashia); 800 m (Bishkek, Kyrgyzstan) and 2640 m (Bogota, Colombia). PK activity, PKLR transcript levels, and ATP decreased at 2640 m compared to 800 m, while p50 increased at 2640 m. PKLR transcript levels correlated with PK activity and ATP and inversely correlated with p50. PK activities also correlated with PKLR transcript levels and ATP and inversely correlated with p50. At 2640 m, PK activity was lower and p50 levels were higher in those with the enriched PKLR haplotype. These results demonstrate that increasing altitude decreases PK activity, resulting in increasing p50 providing a molecular basis for the previously reported improvement of oxygen delivery at high altitude (PMID:17394415).To study the roles of HIFs in the regulation of PKLR gene expression, we also collected samples from Chuvash polycythemia (CP) homozygotes and Chuvash controls. CP homozygotes have a mutation in the VHL gene, a negative regulator of HIFs, that results in augmented HIF levels. CP homozygotes had lower PKLR mRNA in reticulocytes, PK activity, and PKR protein levels in red blood cells compared to controls, while their 2,3 DPG levels were higher. These data confirm that PKLR expression levels are negatively regulated by HIFs.Our findings demonstrate that individuals in high altitudes have lower PKLR transcript levels and PK activity, resulting in high 2,3DPG and p50 which shifts the hemoglobin dissociation curve to right. This decreases affinity of hemoglobin for oxygen, which improves tissue oxygen delivery and as such is another mechanism in Tibetans that protects from high altitude polycythemia. We also demonstrate that HIFs negatively regulate PKLR expression, leading to better oxygen release from hemoglobin at high altitude. DisclosuresKosinski:Agios: Employment, Equity Ownership. Kung:Agios: Employment, Equity Ownership. van Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A comparison of blood nitric oxide metabolites and hemoglobin functional properties among diving mammals

The ability of marine mammals to hunt prey at depth is known to rely on enhanced oxygen stores and on selective distribution of blood flow, but the molecular mechanisms regulating blood flow and oxygen transport remain unresolved. To investigate the molecular mechanisms that may be important in regulating blood flow, we measured concentration of nitrite and S-nitrosothiols (SNO), two metabolites of the vasodilator nitric oxide (NO), in the blood of 5 species of marine mammals differing in their dive duration: bottlenose dolphin, South American sea lion, harbor seal, walrus and beluga whale. We also examined oxygen affinity, sensitivity to 2,3-diphosphoglycerate (DPG) and nitrite reductase activity of the hemoglobin (Hb) to search for possible adaptive variations in these functional properties. We found levels of plasma and red blood cells nitrite similar to those reported for terrestrial mammals, but unusually high concentrations of red blood cell SNO in bottlenose dolphin, walrus and beluga whale, suggesting enhanced SNO-dependent signaling in these species. Purified Hbs showed similar functional properties in terms of oxygen affinity and sensitivity to DPG, indicating that reported large variations in blood oxygen affinity among diving mammals likely derive from phenotypic variations in red blood cell DPG levels. The nitrite reductase activities of the Hbs were overall slightly higher than that of human Hb, with the Hb of beluga whale, capable of longest dives, having the highest activity. Taken together, these results underscore adaptive variations in circulatory NO metabolism in diving mammals but not in the oxygenation properties of the Hb.

Abstract 4230: Visualizing the distribution of metabolites and the efficacy of BIBF1120 on metabolic status of lung cancer derived tumors by imaging mass-spectrometry (MS)

Abstract Background: Imaging MS (Shimadzu CO., LTD.) is a novel technology that can quantitatively visualize the distribution of hundreds of metabolites in the selected areas of tissues. BIBF1120 (gift from Boehrlinger Ingelheim Seiyaku CO., LTD.) is a multiple tyrosine kinase inhibitor, targeting VEGFR, FGFR and PDGFR, and is known as an anti-angiogenic agent. Anti-angiogenic drugs are supposed to induce under-nutrition in tumors. However, no study has visualized the regional differences in the metabolic status of tumors induced by anti-angiogenic agents. Aim: The aims of this study are to visualize the distribution of metabolites in tumors and metabolic status induced by BIBF1120 and to obtain the direct proof that BIB1120 induce under-nutrition status in tumors by Imaging MS. Method: We used lung cancer derived cell lines, namely, PC9, H1975, H3122, A549, H69, and H520. We evaluated distribution of metabolites in tumors derived from xenograft models by Imaging MS. The efficacy of BIBF1120 was evaluated by MTS assay and mouse xenograft model. We treated the engrafted mice with BIBF1120 or vehicle for 4 weeks and harvested xenograft tumors with isotope labeled glucose injection. Micro-vessel density and percentage of apoptotic cells in the tumors were evaluated by immunohistochemistry using anti-CD34 antibody and anti-cleaved caspase 3 antibody, respectively. The effect of BIBF1120 on metabolic status in xenograft tumors was evaluated by imaging MS. Result: Imaging MS revealed heterogeneity of metabolites distribution in the tumors before BIBF1120 treatment. In areas with rich ATP, many viable cells were seen with abundant glutathione, an anti-oxidant metabolite and glutamate, a metabolite derived from oxidative phosphorylation. Otherwise, other areas with rich lactate, a metabolite derived from glycolysis pathway, have less ATP and glutathione level compared with the areas with rich ATP. BIBF1120 inhibited growth of all of the xenograft tumors tested, although it did not directly show inhibitory effect for proliferation rate of those cells in vitro. BIBF1120-treated tumors exhibited significantly lower micro-vessel density compared to the vehicle-treated tumors (p&amp;lt;0.05) and reduced 2-3 DPG (a maker of blood flow), glucose, and ATP levels. Lactate level was increased in BIBF1120-treated tumors. Imaging MS with isotope labeled glucose injection revealed that BIBF1120 treatment induced inactivation of oxidative phosphorylation pathway and activation of glycolysis pathway, especially in the peripheral areas of tumors. Conclusion: We successfully visualized the heterogeneous distribution of metabolites in the tumors, and the effect of BIBF1120 on human lung cancer derived tumors. This is the first report to visualize the distribution of metabolites in engrafted tumors and the efficacy of BIBF1120 on the nutrition status of tumors. Citation Format: Daisuke Arai, Hiroyuki Yasuda, Kenzo Soejima, Shizuko Kagawa, Junko Hamamoto, Shigenari Nukaga, Katsuhiko Naoki, Katsura Emoto, Yuki Sugiura, Makoto Suematsu, Tomoko Betsuyaku. Visualizing the distribution of metabolites and the efficacy of BIBF1120 on metabolic status of lung cancer derived tumors by imaging mass-spectrometry (MS). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4230.

Cod Liver Oil Supplementation Effects on 2,3‐ Diphosphoglyceric Acid Blood Levels in Atherogenic Rabbits

2,3 Diphosphoglyceric Acid (2,3‐DPG) promotes release of oxygen from hemoglobin in the blood of higher animals, and may reduce atherogenic plaque development in hyercholesterolemic rabbits. Eicosapentanoic acid (EPA) converts arachidonic acid in cell membranes to Prostaglandin I‐3, which inhibits platelet aggregation and reduces blood clot formation. Atherogenesis and increased blood vascular fibrin clot formation can be induced in rabbits by feeding a diet containing 2% cholesterol for a month. Blood clot formation at the intimal surface in the aorta reduces oxygen availability which leads to lipid infiltration and plaque development. The effect of feeding atherogenic rabbits cod liver oil (CLO) containing 10% EPA, was studied here to determine its effect on blood levels of 2,3 Diphosphoglyceric Acid (2,3‐DPG).For a month, 12 white, New Zealand strain, male rabbits were fed atherogenic diet (AD) of 92% Purina Rabbit Chow(PRC), 6% Corn Oil and 2% Cholesterol. In the next 8 weeks, 6 rabbits were fed a control diet (CD) of PRC and 6 continued to be fed AD. Half of the CD and AD groups receied 0.8ml CLO daily. Blood 2,3‐DPG means after 2 weeks were: 1.51ug/ml in (CD ) vs 1.75 in the (CD + CLO) and 1.39 in ( AD) vs 1.81 in ( AD+ CLO) animals. After 8 weeks the means were 2.12 (CD) vs 2.35 (CD+CLO), and 1.51 (AD) vs 1.82 (AD+ CLO). The ( CD+CLO ) animals had the highest 2,3 DPG levels of all groups and the least aortal lipid infiltration. (AD+CLO) 2,3 DPG levels were significantly higher than in (AD) controls and they had lower lipid infiltration in their aortae. Blood plasma cholesterol levels in the AD groups were significantly lower in the ( AD+CLO ) vs the (AD) control rabbits.

Red cell storage in E‐Sol 5 and Adsol additive solutions: paired comparison using mixed and non‐mixed study designs

If transfusion of older stored red cells is found to negatively affect clinical outcome, one possible alternative to shortened outdate is the use of new additive solutions (AS) that ameliorate the storage lesion. Erythro-Sol (E-Sol), a previously developed next-generation AS, has been reformulated into E-Sol 5, which is compatible with current anticoagulants and AS volumes. The effect of E-Sol 5 on red cells during storage compared to current AS has not been reported. Paired, ABO-matched whole-blood units were collected into CPD anticoagulant, pooled, split and processed into plasma and red cell units with either 110ml of Adsol or 105ml of E-Sol 5 within 8h of collection. In Study 1, paired units in E-Sol 5 and Adsol were sampled on Day 0 and every 7days up to Day 42 (n=10). In Study 2, paired units in E-Sol 5 and Adsol were sampled only on Day 0 and Day 42 (n=10). In Study 1, 2,3 DPG levels were maintained until Day 28 in E-Sol 5 units and Day 14 in Adsol units. ATP levels were higher in E-Sol 5 units until Day 21, after which they were comparable between the two groups. In both studies, metabolic activity was greater in E-Sol 5 units with respect to glucose consumption and lactate production. Morphology scores were higher, and haemolysis and microparticles generated were lower in E-Sol 5 vs. Adsol units. Weekly mixing of units lowered haemolysis and microparticle levels and increased potassium content on Day 42 in both additive solutions. Regardless of whether units are mixed weekly or are stored non-mixed, E-Sol 5 slows the progression of the red cell storage lesion and improves the overall in vitro quality of RBC throughout storage.

Diurnal Blood Pressure Variations Are Associated with Changes in Distal–Proximal Skin Temperature Gradient

It is generally assumed that skin vascular resistance contributes only to a small extent to total peripheral resistance and hence to blood pressure (BP). However, little is known about the impact of skin blood flow (SBF) changes on the diurnal variations of BP under ambulatory conditions. The main aim of the study was to determine whether diurnal patterns of distal SBF are related to mean arterial BP (MAP). Twenty-four-hour ambulatory measurements of BP, heart rate (HR) and distal (mean of hands and feet) as well as proximal (mean of sternum and infraclavicular region) skin temperatures were carried out in 51 patients (men/women = 18/33) during a 2-d eye hospital investigation. The standardized ambulatory protocol allowed measurements with minimal interference from uncontrolled parameters and, hence, some conclusive interpretations. The distal minus proximal skin temperature gradient (DPG) provided a measure for distal SBF. Individual cross-correlation analyses revealed that the diurnal pattern of MAP was nearly a mirror image of DPG and hence of distal SBF. Scheduled lunch and dinner induced an increase in DPG and a decline in MAP, while HR increased. Low daytime DPG (i.e. low distal SBF) levels significantly predicted sleep-induced BP dipping (r = −.436, p = .0014). Preliminary path analysis suggested that outdoor air temperature and atmospheric pressure may act on MAP via changed distal SBF. Changes in distal SBF may contribute to diurnal variation in MAP, including sleep-induced BP dipping and changes related to food intake. This finding might have an impact on individual cardiovascular risk prediction with respect to diurnal, seasonal and weather variations; however, the underlying mechanisms remain to be discovered.

Guidelines on the use of irradiated blood components prepared by the British Committee for Standards in Haematology blood transfusion task force

Search terms included: Transfusion-associated graft-versus-host disease, Transfusion-associated graft-versus-host, TA-GvHD. The last guideline covering this topic was published in 1996 (British Committee for Standards in Haematology (BCSH) Blood Transfusion Task Force, 1996. The writing group produced the new draft guideline, which was subsequently revised by consensus by members of the Haemato-oncology and Blood Transfusion Task Forces of the BCSH. The guideline was then reviewed by a sounding board of approximately 100 UK haematologists, the BCSH and the committee of the British Society for Haematology and amended, again by consensus. Criteria used to quote levels and grades of evidence are according to the GRADE system (Guyatt et al, 2006). Strong recommendations (grade 1, ‘recommended’) are made when there is confidence that the benefits either do or do not outweigh the harm and burden and costs of treatment. Where the magnitude of benefit or not is less certain a weaker grade 2 recommendation (‘suggested’) is made. Grade 1 recommendations can be applied uniformly to most patients whereas grade 2 recommendations require judicious application. The quality of evidence is graded as A (high quality randomized clinical trials), moderate (B) or low (C). This publication reports the key recommendations of the Writing Group. It is also accessible at http://www.bcshguide lines.com.

Double red cell concentrates -in vitro quality after delayed refrigeration

Automated collection of red cell concentrates (RCC) presents a number of potential advantages to donors, blood services and recipients, and allows the collection of finished components from sites that are remote from a blood centre. However, data are lacking on how long the collected RCC may be stored at ambient temperature prior to their final storage at 4 °C. In this study, the Haemonetics Cymbal device was used to collect RCC using citrate, phosphate and dextrose (CPD-50) anticoagulant. A total of 10 procedures each yielded two leucodepleted RCC in saline, adenine, glucose and mannitol (SAGM) additive solution. One of each pair of RCC was kept warm in an insulated transport bag for 8 h and the other for 6 h. In vitro assessments of the quality of the RCC were made during subsequent 42-day storage of the RCC at 2-6 °C, and compared with reference data. All collected RCC were within UK and European limits for volume, haematocrit and haemoglobin content. Haemolysis was within specification at Day 42 and was no different in RCC held warm for 6 or 8 hours, but tended to be higher than reference data from whole blood derived RCC. ATP, 2,3 DPG and supernatant potassium levels were all similar in RCC held warm for 6 or 8 hours and reference data. We conclude that the Cymbal device may be used to collect two RCC in SAGM, and the in vitro assessment indicates that RCC may be stored without refrigeration for up to 8 h following collection, prior to final storage at 4 °C.

Response to Zimmermann et al

Dear Editor, We appreciate the opportunity to respond to Zimmermann et al. They refer to publications by Vamvakas [1] and Heuft et al. [2]. Both base their conclusion on mathematical models. Vamvakas presents calculations for additional transfusion transmitted infections (TTI) contracted through platelet transfusions in the United States (US) if pooled platelet concentrate (PPC) would completely replace singledonor apheresis platelet concentrates (APC)[1]. These calculations are based on assumptions which do not all apply for Europe. The calculation is based on the risk of TTI in the US today (1 per 2 135 000 donations for HIV, 1 per 1 935 000 for HCV and 1 per 280 000 for HBV [1]). For the German Red Cross Blood Donor Services, the residual risk for TTI is substantially lower (data 1997–2005): 1 per 4 300 000 donations for HIV, 1 per 10 880 000 for HCV and 1 per 360 000 for HBV [3]. This analysis does not account the effect of anti-HBc testing, which became mandatory in Germany since 2006 and will most likely reduce the risk for HBV. The ‘worst-case scenario’ of Vamvakas who carefully confines the conclusions to the United States assumes that a PPC contains platelets from six whole blood (WB) donations [1]. In many European countries, PPC is pooled from five or four donations. The risk calculation is substantially different if assuming six or four donor exposures per PPC [1]. A significant proportion of APC represents split products so that an infected donor can infect two or even three recipients by one donation and in addition further recipients by subsequent donations during the ‘window period’ because of the potentially short interval of APC donations [4]. A model on GBV-C virus infections takes into account that pooling of a GBV-C RNA positive buffy coat (BC) with a BC containing a GBV-C neutralizing antibody could lead to a non-infectious PPC. A careful sensitivity analysis demonstrates that the number of theoretical GBV-C transmissions depends on the assumed frequency of neutralizing antibodies in the donor population [2]. Many assumptions affect the results of mathematical models and delimit their conclusions [4]. Zimmermann et al. argue that the preparation of PPC affects the quality of red blood cell concentrates (RCC) resulting ‘in a remarkable proportion of underfilled RCC’. The binding German Haemotherapy Guidelines [5] and the ‘Guide to the preparation, use and quality assurance of blood components’ [6] stipulate that at least 95% of the leucocyte-depleted RCC must contain ‡ 40g haemoglobin ⁄unit. The mean haemoglobin of RCC produced by our Blood Service using RCC filtration after BC-removal is 56AE4 g, and only 0AE2% of RCC had haemoglobin content below 40 g ⁄unit, and there is no difference when comparing haemoglobin content in RCC produced from donations without or with concomitant preparation of a PPC. For our Blood Service, we can decline that preparation of PPC by the BC method results in a remarkable proportion of underfilled RCC – and this applies also to other blood services obliged to meet regulatory requirements. Zimmermann et al. refer to the loss of 2,3-DPG during overnight storage at 22 C [7]. Several aspects need to be considered in interpretation of this surrogate parameter: Rapid cooling slowed the loss of 2,3-DPG levels but 2,3DPG levels were undetectable by day 21 irrespective of the initial storage temperature [7]. Wilsher et al. [7] compared processing within 8h versus storage for 24 h at 22 C. In practice, the actual interval between donation and start of processing in most institutions is shorter than 24 h even if overnight storage at 22 C is involved. It has been shown that 2,3-DPG levels return to near normal levels after transfusion [8]. The clinical significance of the observed difference needs to be studied further. Overnight storage at 22 C can increase the efficiency of separation of platelets from WB donations and can reduce bacterial contamination. Zimmermann et al. argue that the processing method affects the levels of platelet-derived cytokines in RCC [9]. It is important to take into account all details of the processing. A high concentration of platelet-derived cytokines was measured in PC prepared with the dock-in Sepacell RBC filter or with platelet-saving WB filters before removing plasma or platelet-rich plasma. In this study, the plasmareduced RCC (still containing platelets) were held overnight at 4 C. Neither a storage at 4 C nor a storage overnight after removal of plasma nor the use of platelet-saving WB filters can be considered as standard procedure for PPC Vox Sanguinis (2010)

Treatment with hydroxyurea in a patient compound heterozygote for a high oxygen affinity hemoglobin and β‐thalassemia minor

Compound heterozygotes for β-thalassemia and high oxygen affinity hemoglobin (Hb) have been documented, but experience in the management of such rare cases is minimal. Although hydroxyurea (HU) has never been used in a heterozygote with high oxygen affinity Hb and β-thalassemia, we hypothesized that it would decrease erythrocytosis through a lowered production of abnormal cells and increase of P 50 by induction of fetal hemoglobin (HbF). We present the case of a patient with compound high oxygen affinity Hb mutation with β-thalassemia. PCR analysis revealed combined Hb Regina and IVS1-110 G/A mutations. Treatment with HU caused a decrease in Ht (61.1% to 38.6%) and erythrocyte volume (74.87 mUkg to 40.65 mUkg), as well as an increase in P 50 (6 mmHg to 10 mmHg ) and HbF level (3.6% to 29.8%) at 12-month follow-up. Effects of HU on 2,3 DPG and HbNO levels did not appear to be predictable. Corroboration with other cases is needed to establish solid evidence on the clinical efficacy of HU in this population.

The effects of additive solution pH and metabolic rejuvenation on anaerobic storage of red cells

Red cell (RBC) storage can be extended to 9 weeks under anaerobic or alkaline conditions. Simultaneous use of these approaches has not provided additive benefit. Our objective was to determine whether anaerobic storage with acidified additive solution (AS) coupled with metabolic rejuvenation might further improve the benefits of anaerobic storage. RBC storage in AS with a pH value of 6.5, 7.4, or 8.3 in aerobic or anaerobic conditions was examined using a panel of in vitro biochemical and RBC markers. RBC rejuvenation during cold storage was also evaluated. A randomized crossover radiolabeled recovery study (eight subjects) evaluated anaerobic RBC storage using AS65 with cold rejuvenation for up to 16 weeks of storage. Adenosine triphosphate (ATP) and diphosphoglycerate acid (DPG) were better maintained in anaerobic storage than in aerobic storage. Acidic or neutral AS preserved ATP concentration better, while a neutral or basic pH AS favored maintenance of DPG levels at higher levels for a longer period. AS pH had less of an effect on exposure of phosphatidylserine (PS), vesicle protein release, and hemolysis. Rejuvenation of RBCs during cold, anaerobic storage resulted in increases in ATP and DPG levels and a reversal of PS exposure. Anaerobic storage of RBCs in pH 6.5 AS rejuvenated at 7 weeks of storage yielded RBC 24-hour recoveries of 77.3 +/- 12.5 percent after 10 weeks' storage time. After a second rejuvenation at Week 11, six subjects' units demonstrated a recovery of 75.9 +/- 7.3 percent at 12 weeks of storage. Extended RBC storage may be achieved using anaerobic conditions combined with low-pH AS and rejuvenation during storage.

Red cell pyruvate kinase deficiency in neonatal jaundice cases in India

Pyruvate Kinase (PK) deficiency is the most common enzymopathy of the glycolytic pathway in erythrocytes. It constitutes one of the common causes of hereditary non-spherocytic hemolytic anemia. The aim of this study was to screen newborns in India for pyruvate kinase (PK) deficiency in relation to unconjugated hyperbilirubinemia. Laboratory investigations done included complete blood counts, reticulocyte counts, direct and indirect bilirubin, assay of G6PD and PK activity, ATP and 2,3 DPG levels. All variables were studied in 50-cord blood samples from normal deliveries and 218 neonates with hyperbilirubinemia. 7 of the 218 cases of neonatal jaundice were PK deficient with 30-40% reduction in PK activity. These cases also had a 3-4-fold increase in 2,3 DPG:ATP ratios, which is one of the additional indicators for PK deficiency. Six of the 7 infants had a severe clinical course. This study shows that the prevalence of PK deficiency in Indian neonatal jaundice cases is 3.21%, which is relatively high. This emphasizes the need for screening neonatal hyperbilirubinemia cases in India for PK deficiency.