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HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC ) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral (15) N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15-20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R(2) = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c.

Increased frequency and risk of infection is one of the well described complications of sickle cell anemia (SCA). Dietary supplementation in children with SCA and growth retardation improved growth and decreased incidence of infection. We investigated the impact of a high protein diet on weight gain, hematological profile, and immune cytokine levels in the Berkeley model of SCA, 16 of which were randomized to either regular mouse diet with 20% of calories from protein ( n = 8) or a test feed with 35% of calories from protein ( n = 8). Control mice (C57BL/6, n = 16) were correspondingly randomized, and were all feed ad libitum for three months with actual intake estimated by subtracting the weight of gnaw waste from that of the feed given. Blood was collected at sacrifice by cardiac puncture and plasma levels of T helper cell 1 (TH1) and TH2 associated cytokines were measured using a multiplex antibody immobilized bead assay. SCA mice receiving the 35% protein diet had modest improvements in weight, red blood cell count, and hemoglobin level, with a slight decrease in reticulocyte count compared with SCA mice on the regular mouse diet. Furthermore, they also had significantly higher plasma levels of cytokines tumor necrosis factor (TNF)-α ( P = 0.02), interferon (IFN)-γ ( P = 0.01), interleukin 10 (IL-10; P = 0.02), and IL-4 ( P = 0.02) compared with those that received the 20% protein diet. We conclude that providing additional protein calories to transgenic SCA mice increased the plasma levels of acute inflammatory cytokines associated with immune response to infection, which might partly explain decreased episodes of infection observed among supplemented children with SCA.

Sickle cell disease is associated with hypermetabolism and a consequent shortage of substrates for normal growth and healthy immune response. The protein:energy ratio is a major determinant of dietary adequacy; the requirement for optimal growth of control mice is 20% of energy from dietary protein. This study investigated the efficacy of increased dietary protein for improving weight gain and reducing inflammation in the Berkeley sickle cell mouse model (S). The study examined the effect of diet on weight gain and circulating levels of 2 inflammatory proteins, C-reactive protein (CRP), and cytokine interleukin-6 (IL-6). Male C57BL/6 (C) control (n = 8) and S mice (n = 8) were randomized at weaning to 40 d of isoenergetic diets containing 20% (normal) and 35% (high) of energy from protein (C20, C35, S20, S35), replacing dextrin. Rate of weight gain was calculated and plasma CRP and IL-6 concentrations determined by ELISA. Liver mRNA expression of these proteins was measured by real-time PCR and L-arginase by colorimetric assay. S35 mice tended to gain weight more rapidly than S20 mice (P = 0.06) and more rapidly than C35 mice (P < 0.01). Circulating CRP and IL-6 levels were also lower in S35 mice than in S20 mice (P < 0.05), as was liver CRP mRNA expression (P < 0.01). These results demonstrate that introducing a high protein diet at weaning attenuates the steady-state inflammation in this S mouse model. Dietary L-arginine availability was investigated as a possible mechanism for increased nitric oxide production and consequent reduced inflammation.

Summary Background & aims Current self-report methods to monitor dietary intake are often unreliable. As part of a dietary intervention study, we investigated whether adding a common food flavor (vanillin) to test diets and measuring the major metabolic end product vanillic acid in urine, could provide assessment of compliance with dietary supplements. Methods After baseline urine was collected 10 subjects (6 control and 4 study patients) consumed 1.3 g of vanillin in a liquid test meal as the last food at bedtime and collected the first morning urine. Next a kinetic excretion study was performed in which 6 controls consumed a vanillin spiked drink with continued urine sampling at 30-min intervals for 5 h. Vanillic acid concentrations were measured by reverse phase high-performance liquid chromatography. Results The test diet was consumed just before bedtime; the first morning void vanillic acid concentration gave a reliable indication of compliance (3 males 0.224±0.041 and 3 females 0.290±0.099 mg/ml; mean±SD). Thirty-minute sampling of vanillic acid excretion for 6 controls was maximal 1 h after the test diet, returning to baseline after 4 h. Conclusion Vanillin is a useful, inexpensive and non-toxic biochemical marker for confirming compliance with experimental diets.

Sickle cell anemia (HbSS) includes chronic inflammation, but the origin is unclear. We hypothesized that in stable HbSS patients the inflammation was associated with hypermetabolism. We compared selected hypermetabolic and key immunomodulator indicators in HbSS versus control children and examined associations between measures of hypermetabolism and inflammation. Twelve fasting asymptomatic HbSS children 6-12 years and 9 controls matched for age, gender and fat mass (FM) were studied. Proportional reticulocyte count (retic%) and resting energy expenditure (REE) represented hypermetabolism, and C-reactive protein (CRP) indicated inflammation. Proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), chemokine monocyte chemoattractant protein-1 (MCP-1), and energy balance cytokine leptin were measured. Methods were indirect calorimetry, enzyme-linked immunosorbent assay, and radioimmunoassay. Statistical analysis included simple correlation and regression analysis. REE (51 +/- 6 vs. 43 +/- 12 kcal/kg per fat-free mass (FFM), mean +/- SD), retic% (12 +/- 4 vs. 0.7 +/- 0.3%), CRP (5 +/- 3 vs. 0.3 +/- 0.4 mg/liter), and IL-6 (71 +/- 40 vs. 20 +/- 7 pg/ml) were significantly higher for HbSS than controls (P0.05). Conversely, leptin (0.1 +/- 0.1 vs. 2 +/- 1 microg/liter per kgFM) and MCP-1 (34 +/- 5 vs. 41 +/- 4 pg/ml) were significantly lower for the HbSS subjects (P0.01). TNF-alpha was not significantly different. There were no significant associations between REE or retic% and any cytokine measured. However, CRP was significantly associated with REE in HbSS (r = 0.8, P = 0.003) and an important predictor of REE/FFM. We provide new evidence for low circulating levels of inflammatory chemokine MCP-1 in stable HbSS children, confirm mostly low cytokine levels, inflammation, and hypermetabolism and demonstrate association of hypermetabolism with inflammation via CRP but not via cytokines.

Key pathophysiology of sickle cell anaemia includes compensatory erythropoiesis, vascular injury and chronic inflammation, which divert amino acids from tissue deposition for growth/weight gain and muscle formation. We hypothesised that sickle mice maintained on an isoenergetic diet with a high percentage of energy derived from protein (35 %), as opposed to a standard diet with 20 % of energy derived from protein, would improve body composition, bone mass and grip strength. Male Berkeley transgenic sickle mice (S;n8–12) were fed either 20 % (S20) or 35 % (S35) diets for 3 months. Grip strength (BIOSEB meter) and body composition (dual-energy X-ray absorptiometry scan) were measured. After 3 months, control mice had the highest bone mineral density (BMD) and bone mineral content (BMC) (P P = 0·043) attributed this difference to genotype (P = 0·025) and a trend in type of diet (P = 0·067).l-Arginine (l-Arg) supplementation of the 20 % diet was explored, as a possible mechanism for improvement obtained with the 35 % diet. Townes transgenic sickle mice (TS;n6–9) received 0·8, 1·6, 3·2 or 6·4 %l-Arg based on the same protocol and outcome measures used for the S mice. TS mice fed 1·6 %l-Arg for 3 months (TS1.6) had the highest weight gain, BMD, BMC and lean body mass compared with other groups. TS3.2 mice showed significantly more improvement in grip strength than TS0·8 and TS1.6 mice (P l-Arg as part of the mechanism.

We developed a method to measure hemoglobin synthesis rate (SynHb) in humans, assuming that free glycine in the red blood cell (RBC) represents free glycine in bone marrow for hemoglobin synthesis. The present rat study examines this assumption of the method and quantifies SynHb in rats. Sprague-Dawley rats (n = 9) were studied, [2-(13)C]glycine was intravenously infused over 24 h (2.5 mg kg(-1) h(-1)), blood was drawn for glycine and heme isolation, and bone marrow was harvested for glycine isolation. Isotopic enrichments of glycine and heme were measured, fractional hemoglobin synthesis rate (fSynHb% day(-1)) was calculated, and from this a value for SynHb (mg g(-1) day(-1)) was derived. Mean body weight was 446 +/- 10 g (mean +/- SE) and hemoglobin concentration was 14 +/- 0.5 g dl(-1). At 24 h, the mean isotopic enrichment, atom percentage excess (APE), of the RBC free glycine (1.56 +/- 0.18 APE) was similar to the bone marrow (1.68 +/- 0.15 APE). The rate of incorporation of (13)C into heme increased over time from 0.0004 APE/h between 6 and 12 h, to 0.0014 APE/h between 12 and 18 h, and 0.0024 APE/h between 18 and 24 h. Consequently, fSynHb (1.19 +/- 0.32, 2.92 +/- 0.66, and 4.22 +/- 0.56% day(-1), respectively) and SynHb (0.11 +/- 0.03, 0.28 +/- 0.05, and 0.42 +/- 0.05 mg g(-1) day(-1), respectively) showed similar patterns over the 24-h study period. We conclude that (1) enrichment of free glycine in the circulating RBC approximates enrichment of bone marrow free glycine for heme formation and (2) this pattern of hemoglobin synthesis rate is reflecting the characteristic release and gradual maturation of reticulocytes in the circulation.

Background: A higher than normal basal inflammatory state is characteristic of sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA approved drug for SCD. However, HU is a chemotherapy drug and is therefore naturally cytotoxic, often inducing apoptosis. Chronic inflammation in sickle cell patients is invariably associated with injury to the vascular endothelium. Quercetin is a dietary flavonoid found ubiquitously in plants and foods that have anti-oxidative and anti-inflammatory characteristics. Hypothesis: The dietary flavonoid quercetin will decrease cytotoxic effects of Lipopolysaccharide and HU induced cell damage to vascular endothelial cells. Aims: 1. to develop an in vitro inflammatory model simulating chronic baseline inflammation observed in sickle cell disease. 2. to test the effect of the physiological dose of hydroxyurea (HU), on the inflammatory model. 3. to examine the role of quercetin (QCT), a dietary flavonoid with anti-oxidative and anti-inflammatory characteristics, for reducing the inflammation. Methods: Lipopolysaccharide (LPS) was used to induce inflammation in immortalized mouse aortic endothelial cells (iMAECs), providing an in vitro model of inflamed endothelial cells. The cells were exposed to LPS throughout the entire experiment. Interventions included treating the LPS exposed cells with QCT, HU, or QCT + HU over 50 hours. The 50-hour period included 24 hours of varying treatments, followed by two hours of hypoxic exposure and then 24 hours under normal aerobic exposure. Untreated cells (controls) provided a comparison. Data analyses included comparisons of control cells with the inflammatory model, and pairwise comparisons between the inflammatory model and the different treatments. In this experiment lactate dehydrogenase (LDH) was measured by colorimetric assay, as an indication of cell damage. Results: At the end of the experiment, the LDH level for the inflammatory model was significantly higher than LDH for the control cells (P = 0.0005) fig 1. Treatment with 30 micromoles QCT gleaned a trend toward reduced LDH compared with the inflammatory model (p = 0.1) fig 2. LDH was significantly higher after treatment with 100 micromoles HU compared with the inflammatory model (p = 0.0005) Fig 3. However, LDH was significantly reduced after treatment with a combination of 30 micromoles QCT/100 micromoles HU, compared with 100 micromoles HU alone (p = 0.0008) fig 3. Conclusions: These results suggest that quercetin may be effective against vascular endothelial cell damage for iMAECs in vitro. It also shows promise in preventing HU-induced cytotoxicity, which was a surprising finding from these results. This latter finding is interesting, and should be given more consideration, since HU is the only FDA-approved drug for treating sickle cell patients, and its use is rapidly increasing. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

OBJECTIVE. The purpose of this study was to quantitate the derangements in intermediary carbohydrate metabolism and oxygen use in severely septic patients in comparison with healthy volunteers. SUMMARY BACKGROUND DATA. It commonly has been assumed that the development of lactic acidosis during sepsis results from a deficit in tissue oxygen availability. Dichloroacetate (DCA), which is known to increase pyruvate oxidation but only when tissue oxygen is available, provides a means to assess the role of hypoxia in lactate production. METHODS. Stable isotope tracer methodology and indirect calorimetry was used to determine the rates of intermediary carbohydrate metabolism and oxygen use in five severely septic patients with lactic acidosis and six healthy volunteers before and after administration of DCA. RESULTS. Oxygen consumption and the rates of glucose and pyruvate production and oxidation were substantially greater (p < 0.05) in the septic patient compared with healthy volunteers. Administration of DCA resulted in a further increase in oxygen consumption and the percentage of glucose and pyruvate directed toward oxidation. Dichloroacetate also decreased glucose and pyruvate production, with a corresponding decrease in plasma lactate concentration. CONCLUSIONS. These findings clearly indicate that the accumulation of lactate during sepsis is not the result of limitations in tissue oxygenation, but is a sequelae to the markedly increased rate of pyruvate production. Furthermore, the substantially higher rate of pyruvate oxidation in the septic patients refutes the notion of a sepsis-induced impairment in pyruvate dehydrogenase activity.

Urea kinetics (urea-N production-P, excretion-E, hydrolysis-H, recycling-R and retention-S) were measured in 7 healthy adults consuming a standard diet compared with 4 fasted for 24 and/or 96 h, using primed/intermittent doses of [(15)N (15)N]-urea and mass spectrometry. Standard values were P = 196, E = 132, H = 65, R = 13 and S = 51, mgN/kg/day. After 24 h fasting all urea kinetics were reduced, and P and H were significantly reduced compared with the standard diet (p0.01 and0.05 respectively). After 96 h fasting, urea kinetics returned to standard values (P = 187, E = 136, H = 51, R =13 and S = 38, mgN/kg/day), although nitrogen intake was significantly lower (p0.001). Relative urea excretion (E/P) was 67%, standard diet, and 75% after fasting. Consequently H/P was slightly reduced from 33 to 25%. S/P was 26%, standard diet, 15% after 24 h and 20% after 96 h fasting, suggesting increased urea-N retention with prolonged fasting. These results imply a slight temporary shift towards increased nitrogen excretion at 24 h and subsequent return to the kinetics of the fed state after 96 h. Urea-N retention increases with prolonged fasting.

Sickle cell anemia (SCA) associated cerebrovascular disease includes vascular remodeling, abnormal cerebral blood flow (CBF) and infarction. We studied the relationships between plasma brain derived neurotropic factor (BDNF), platelet derived growth factors (PDGF-AA and -AB/BB) and high trans-cranial Doppler (TCD) velocity, an indication of CBF velocity. Baseline plasma samples from 39 children (19 SCA with abnormal/high TCD [SATCD], 13 SCA with normal TCD [SNTCD] and 7 healthy non-SCA), were assayed for BDNF, PDGF-AA and – AB/BB plus 11 other cytokines. The sensitivity, specificity and usefulness of these biomarkers for prediction of stroke incidence was investigated. All subject groups were of similar age and gender distribution. Mean BDNF was significantly higher among SATCD than SNTCD (p=0.004) as was mean PDGF-AA (p=0.001). Similarly, mean PDGF-AA was higher among SCA subjects who developed stroke than for those who did not (p=0.012). Elevated BDNF and PDGF-AA were both associated with severity of anemia. Elevated BDNF and PDGF-AA were good predictors of the presence of abnormally high CBF velocity, and PDGF-AA predicted stroke development. Stroke incidence and high TCD velocity were associated with elevated BDNF and PDGF-AA. These findings suggest a role for BDNF and PDGF-AA in the patho-physiological mechanism of cerebrovascular disease in SCA.

C-X-C motif chemokine 10 (CXCL10) also known as interferon γ-induced protein 10 kDa (IP-10) or small-inducible cytokine B10 is a cytokine belonging to the CXC chemokine family. CXCL10 binds CXCR3 receptor to induce chemotaxis, apoptosis, cell growth and angiostasis. Alterations in CXCL10 expression levels have been associated with inflammatory diseases including infectious diseases, immune dysfunction and tumor development. CXCL10 is also recognized as a biomarker that predicts severity of various diseases. A review of the emerging role of CXCL10 in pathogenesis of infectious diseases revealed diverse roles of CXCL10 in disease initiation and progression. The potential utilization of CXCL10 as a therapeutic target for infectious diseases is discussed.

The pathogenesis of sickle cell disease (HbSS), which has numerous complications including stroke, involves inflammation resulting in alteration of plasma inflammatory protein concentration. We investigated HbSS children with abnormal cerebral blood flow detected by trans-cranial Doppler ultrasound (TCD) who participated in multi-center stroke prevention (STOP) study, to determine if plasma inflammatory protein concentration is associated with the outcome of stroke in the STOP study. Thirty-nine plasma samples from HbSS participants with elevated TCD who had no stroke, HbSS-NS (n=13) or had stroke, HbSS-S (n=13), HbSS steady-state controls (n=7) and controls with normal hemoglobin, HbAA (n=6), were analyzed simultaneously for 27 circulating inflammatory proteins. Logistic regression and receiver operating characteristics curve analysis of stroke on plasma inflammatory mediator concentration, adjusted for age and gender, demonstrated that interleukin-1β (IL-1β) was protective against stroke development (HbSS-NS = 19, 17–23, HbSS-S = 17, 16 – 19 pg/mL, median and 25th–75th percentile; Odds ratio = 0.59, C.I. = 0.36 – 0.96) and was a good predictor of stroke (area under curve = 0.852). This result demonstrates a strong association of systemic inflammation with stroke development in HbSS via moderately increased plasma IL-1β concentration, which is furthermore associated with a decreased likelihood of stroke in HbSS.

Hypercoagulability in sickle cell disease (SCD) is associated with multiple SCD phenotypes, association with stroke risk has not been well described. We hypothesized that serum levels of biomarkers of coagulation activation correlate with high transcranial Doppler ultrasound velocity and decreases with blood transfusion therapy in SCD patients. Stored serum samples from subjects in the Stroke Prevention in Sickle Cell Anemia (STOP) trial were analyzed using ELISA and protein multiplexing techniques. 40 subjects from each treatment arm (Standard Care [SC] and Transfusion [Tx]) at three time points--baseline, study exit and one year post-trial and 10 each of age matched children with SCD but normal TCD (SNTCD) and with normal hemoglobin (HbAA) were analyzed. At baseline, median vWF, TAT and D-dimer levels were significantly higher among STOP subjects than either HbAA or SNTCD. At study exit, median hemoglobin level was significantly higher while median TCD velocity was significantly lower in Tx compared to SC subjects. Median vWF (409.6 vs. 542.9 μg/ml), TAT (24.8 vs. 40.0 ng/ml) and D-dimer (9.2 vs. 19.1 μg/ml) levels were also significantly lower in the Tx compared to the SC group at study exit. Blood levels of biomarkers coagulation activation/thrombin generation correlated positively with TCD velocity and negatively with number of blood transfusions. Biomarkers of coagulation activation/thrombin generation were significantly elevated in children with SCD, at high risk for stroke. Reduction in levels of these biomarkers correlated with reduction in stroke risk (lower TCD velocity), indicating a possible role for hypercoagulation in SCD associated stroke.

Objectives: We hypothesized that an elevated hemoglobin synthesis rate (SynHb) and myocardial oxygen consumption (MVO2) contribute to the excess protein and energy metabolism reported in children with sickle cell anemia. Patients and Methods: Twelve children (6–12 years old) with asymptomatic sickle cell and 9 healthy children matched for age and sex were studied. Measurements were whole-body protein turnover by [1- 13 C]leucine, SynHb by [ 15 N]glycine, resting energy expen- diture by indirect calorimetry and the systolic blood pressure–heart rate product used as an index of MVO2. Protein energy cost was calculated from protein turnover. Statistical analysis included Spearman correlations and partial correlation analyses. Results: Although body mass index was significantly lower for sickle cell versus controls (P < 0.02), children with asymptomatic sickle cell had 52% higher protein turnover (P < 0.0005). Proportional reticulocyte count, SynHb, MVO2 and resting energy expenditure were also significantly higher in children with sickle cell (P < 0.01). Protein turnover correlated significantly with both SynHb (r ¼ 0.63, P < 0.01) and reticulocyte percentage (r ¼ 0.83, P < 0.0001). Partial correlation of these 3 variables showed reticulocyte percentage as the only variable to be significantly associatedwithproteinturnover, even after adjustingforsickle cell anemia (P ¼ 0.03). Partial correlation of log resting energy expenditure on MVO2 was significant, controlling for protein energy cost, sex and age (P ¼ 0.03). Conclusion: These results indicate that metabolic demands of increased erythropoiesis and cardiac energy consumption account for much of the excess protein and energy metabolism in children with sickle cell anemia. JPGN 43:680–687, 2006.

Background. Malignancies enlarge because protein synthesis exceeds the rate of breakdown; however, the specific protein kinetic pattern remains unknown. Determining in vivo protein kinetic rates for a tumor may be useful for quantifying individual responses to a specific therapy. The aim of this study was to assess whether the growth of tumors is related to an increase in protein synthesis or an inhibition of protein breakdown. Methods. Five patients (age, 59 ± 3 years) with adenocarcinoma of the colon undergoing colonoscopy were studied. Tissue protein synthesis and breakdown rates were measured in vivo for both segments of colon cancer and adjacent normal-appearing colonic mucosa by using a primed, continuous infusion of 113C leucine with tissue biopsy and quantitation of regional blood flow by laser Doppler flowmetry. Results. Segments of colon cancer had a significantly (p < 0.05) greater rate of protein synthesis as quantitated by both the fractional rate of protein synthesis (Ca 45.4% ± 5.0% /day versus nl mucosa 35.7% ± 3.1%/day; mean ± SEM) and by the tissue synthesis rate (Ca 69.4 ± 9.0 versus nl mucosa 51.6 ± 5.2 μmol/L leucine/day/100 gm tissue). Regional blood flow was significantly elevated in the cancer (Ca 110.9 ± 5.8 versus nl mucosa 91.2 ± 2.9 ml/min/100 gm), which contributed to commensurate rates of tissue breakdown (Ca 28.6 ± 2.0 versus nl mucosa 28.2 ± 2.4 μmol/L leucine/day/100 gm). Conclusions. These results illustrate that human colon cancers grow in vivo as a result of increases in protein synthesis. Furthermore, increases in regional blood flow limit the rate of tissue protein breakdown of colon cancer, thereby contributing to growth of the malignancy. These findings support the contention that therapeutic strategies aimed at negating this inherent increase in protein synthesis or limiting blood flow may effectively limit the growth of malignancies. This methodology may also provide an index for evaluating the effectiveness of future therapies aimed at reducing tumor growth for individual patients.

In Africa, the natural history of SCD is often assumed to be same to the African Diaspora in the US, Jamaica, Europe or Latin America. Yet the environment can be different, including different pathogen exposure, such as malaria. To help better understand this, over 2000 references were identified using the names of all current or past names of African continent countries and the truncated word sickl$, followed by secondary nested and cross reference searches. Six cases series describing causes of death were identified, representing 182 children (Ndugwa, 1973, Athale, 1994, Koko, 1998, Diagne, 2000, Rahimy, 2003, Van-Dunem, 2007). Gender was reported in 172, 73 were female (42%). Age was reported in 118, 52 were < 5 years (44%). Four studies described some impediment to care or arrival for care in extremis in1/4 to over ½ of patients that died. In Uganda, 9/12 (75%) patient died at home. In Gabon 6/23 (26%) patients died within 4 hours of reaching the hospital and 11/23 (48%) within 24 hours. In Benin 2/10 (20%) died of splenic sequestration diagnosed at home; 38/64 (53%) of patients in Mozambique that died, lived outside of the capital. Causes of death were identified in 146 individuals. These included: fever/sepsis: n=59 (40%), including meningitis: n=15 (10%) and pyelonephritis: n=2 (1%); acute anemia: n=43 (29%), including spleen sequestration: n=28 (19%) and aplastic anemia: n=8 (5%); pain: n=22 (15%); acute chest syndrome/pneumonia: n=18 (12%); CNS: n=8 (5%), including stroke: n=4 (3%), seizure/ coma: n=5 (3%); liver disease: n=5 (3%) including hepatitis: n=3 (2%); Other: n=19 (13%) including wasting/ malnutrition: n=7 (5%), heart failure/cardiomyopathy: n=4 (3%), diarrhea and vomiting: n=3 (2%), transfusion reaction: n=2 (1%). Infectious pathogens were identified in 26, including malaria: n=10 (38%), S. pneumoniae: n=3 (12%), Salmonella: n=2 (8%), H. influenza, Klebsiella and Citrobacter: n=1 (4%) each; viral agents were reported in n=8 (31%) including HBV: n=5 (19%), HIV: n=3 (12%). Reported general population hemoglobinopathy surveys after birth revealed the following Relative Risk (RR) of observing individuals with hemoglobin SS compared to Hardy Weinberg expected frequencies (some age cohorts overlap; Tanzania '56, Benin '09, Burkina Faso '70, Central African Republic'75, Gabon'65/'80, Gambia'56, Ghana '56/‘57/'00/'10, Kenya '04/'10, Malawi '72/'00/'04, Mozambique '86, Nigeria '56/'70/'79/'81/'84/'05, Senegal '69, Sierra Leone '56). Age 0-1 years, total n=2112 observed n=22 (1.0%), expected n=16.5 (0.8%), RR=1.3 (95% CI=0.7,2.5), p=0.441. Age 0-6 years, total n=4078; observed n=39 (1.0%); expected n=40.6 (1.0%); RR=1.0 (95% CI=0.6,1.5), p=0.925. Age 5-19 years, total n=1880; observed n= 5 (0.3%); expected n= 24.8 (1.3%); RR=0.2 (95% CI=0.1,0.5); p Disclosures: No relevant conflicts of interest to declare.

The authors wish to correct the numbers stated in the body and figures of the article titled ‘Plasma BDNF and PDGF-AA levels are associated with high TCD velocity and stroke in children with sickle cell anemia’, Epub June 15th 2012 and published October 1st 2012 in the Cytokine journal [1]. The values for plasma level of biomarkers quoted in the text and also the scales for the graphs/figures should be multiplied by 100 before use or quoting it. This is to reflect the 1:100 dilution that was done to facilitate the assay procedure and in compliance with the manufacturers protocol. This error was an oversight that was not apparent until recently and because it is impossible to correct the original article at this time. This correction does not in anyway change the observation that was made by the investigators in this study. Although this correction does not invalidate the associations, certain investigators might want to quote exact figures and it for this reason that we are publishing this corrigendum for those who will eventually use this article. We apologize for any inconvenience and are available to answer any questions you may have.

Abstract 244 Stroke is a major cause of morbidity and mortality among children with sickle cell anemia (SCA). Children with SCA at risk for stroke can be identified by transcranial Doppler (TCD) ultrasound screening for abnormally high cerebral artery blood flow velocity and strokes can be prevented by chronic packed red blood cell (RBC) transfusion. However, the mechanisms that lead to cerebral vasculopathy and stroke in SCA and that explain the beneficial effects of chronic RBC transfusions in stroke prevention are poorly understood. We have previously shown that pre-treatment serum levels of brain derived neurotropic factor (BDNF) and platelet derived growth factor (PDGF) subtypes, biomarkers of cerebral ischemia and arterial remodeling, were associated with both high TCD velocity and development of stroke. We hypothesized that frequency of RBC transfusion would be associated with altered serum levels of neurodegenerative, inflammatory and angiogenic markers in SCA children with high TCD velocity and tested this hypothesis by assaying levels of these markers in post-STOP serum samples. Frozen serum samples drawn one year after subject's exit from the STOP clinical trial phase were utilized. Given the positive trial results, all but 9 subjects had been on chronic transfusion regimen for at least one year at the time of sample collection. Eighty samples were assayed using multiplex antibody immobilized beads (Millipore Corp, Billerica, MA). The mean fluorescent intensity was measured using the Milliplex xMAP system powered by Luminex (Bio-Rad, Hercules, CA). Ten biologically related neurodegenerative, inflammatory and angiogenic biomarkers were tested. The total number (frequency) of RBC transfusions recorded over the study period (4 years) for each participant was categorized into High (≥ 40), Moderate (20 – 39) or Low (< 20) frequency of transfusion. Median distribution with 10 – 90th percentile of the levels of biomarkers and TCD velocity were expressed using box-plots and the differences in median distribution between groups based on frequency of transfusion was estimated using Kruskal-Wallis test. A principal component analysis (PCA) loading plot was used to demonstrate the biological relationships between the biomarkers, taking into consideration linear correlations and spatial relationships between them. There were no significant differences in the hematological and anthropometric measures between groups. Overall, our result showed that low transfusion frequency was associated with high serum levels of biomarkers and vice versa, despite no significant difference in hemoglobin level between groups. The high frequency transfusion group had lower serum levels of BDNF (p = 0.02), sVCAM-1 (p < 0.001), PDGF-AA (p < 0.001), CCL5 (p < 0.01), tPAI-1 (p < 0.01) and NCAM (p < 0.01) levels compared with the low frequency transfusion group (figure 1 a – e). Although not shown in the figures, the same pattern was observed with TCD velocity which was lower (160, 115.7 – 204.9 cm/s) in the low compared with the high (195, 154 – 272 cm/s) frequency transfusion group. In addition, the medium frequency transfusion group had significantly lower serum sVCAM-1 (p < 0.01) compared with the low frequency transfusion group and higher PDGF-AA (p < 0.01) compared with the high frequency transfusion group. A PCA loading plot (figure 2) shows clustering of the biomarkers that are most closely biologically related, these are also the biomarkers that were significantly affected by the frequency of transfusion. Red blood cell transfusions in the STOP study were associated with reduced serum levels of biomarkers of angiogenesis (PDGF-AA and sVCAM-1), cerebral ischemia/neuronal survival adaptation (BDNF and NCAM) and inflammation (RANTES/CCL5), and this effect was most pronounced in the group with the highest frequency of transfusions (equivalent to most chronic transfusion regimen). This suggests that the protective effects of chronic RBC transfusions on stroke development in children with SCA may be attributable to improved cerebral perfusion, reduced inflammation and down-regulation of hypoxia-induced angiogenic responses that promote arterial remodeling. One or more in this group of biologically-related and relevant markers may be useful for monitoring children with SCA receiving stroke prevention therapies and for designing treatment targets. Disclosures: No relevant conflicts of interest to declare.

Total free-living energy expenditure (TEE) was measured in 9 normal weight controls and 5 obese women using the doubly labeled water (DLW) method. Resting energy expenditure (REE) and the thermic effect of food (TEF) were measured by indirect calorimetry and the energy cost of physical activity (PA) calculated by deduction, in order to quantify the components and identify determinants of free-living TEE. Although REE was quantitatively the major component of TEE in both groups, PA best explained the variability, contributing 76% to the variance in free-living TEE. The obese women had elevated values for TEE (12397 +/- 2565 vs. 8339 +/- 1787 kJ/d, mean +/- SD; p < 0.005), compared with the control women. PA (5071 +/- 2385 vs. 2552 +/- 1452; p < 0.05) and REE (6393 +/- 678 vs. 5084 +/- 259; p < 0.0005) were also raised in the obese, whereas TEF was not significantly different between the groups, accounting for 7.6% of energy expenditure for the obese and 8% for the control subjects. Body weight was the single best determinant of mean daily free-living TEE across both groups. We conclude that PA and body weight are the main determinants of free-living TEE.

Abstract 2123 Little is known about the relative rate of pubertal development among children with different phenotypes of sickle cell disease (SCD). This study proposes, for the first time, to compare the influence of SCD phenotypes on pubertal development (PD) and body mass. We hypothesized that differences in pubertal development and body mass index (BMI) are modulated by SCD phenotype and treatment modality. We therefore conducted a cross-sectional study of SCD children and adolescents aged 8 – 18 years old (yo) to determine the impact of SCD phenotype and treatments on pubertal development (according to sexual maturity rating by Tanner stages, TS) and BMI. Participants were recruited from Children's Healthcare of Atlanta and enrolled after written informed consent was obtained. Subjects' age, height, weight, self-reported TS (via questionnaire), physician-reported TS, and major therapies were recorded. We investigated two major phenotypes, hemoglobin SC disease (SC, n= 16) and sickle cell anemia (SS, n=58). Within the SS group there were three treatment sub-groups, no-treatment (SS, n=18), hydroxyurea (S HU, n=21), and red blood cell transfusion (S Tx, n=19). Since age is associated with achieving a certain level of sexual maturity we divided the participants according to Nelson's Textbook of Pediatrics. A Tanner stage of Early adolescent (10–13 yo) SS females had significantly lower self-reported TS, than those with SC (2±1 vs.3±1, means±SD respectively, p=0.018) or S Tx (3±0, p=0.031). Mid adolescent S HU females had significantly lower self-reported TS than SS (3±1 vs. 5±1, p=0.027) or SC (5±0, p=0.034), reflecting more past SCD complications in the S HU group. One late adolescent male in the Hb S Tx group did not attain Tanner 5. Across all groups, more than 50% of the remaining participants achieved normal PD (Table 1). Investigating BMI (kg/M2) by developmental stages revealed that early adolescent SC females had significantly higher BMI than S HU (23±9 vs. 16±1, p=0.036) and SS females (15±1, p=0.028). For early adolescent S Tx males, BMI was significantly higher than S HU males (19±1 vs.16±1, p=0.034). SS children were more likely than SC to be underweight (11–14%) regardless of treatment, and least likely to be overweight with no treatment (Table 2). These results show that there are differences in level of maturity and BMI based on SCD phenotype and treatment modality. Differences seen in treatments are possibly due to the length of time on treatment modality prior to enrolling in study. Although these differences exist, the majority of participants lie within the normal PD range. Surprisingly, sickle cell disease, which is usually associated with delayed development, did not affect the ability of most these children to reach puberty within the expected timeframe. However, they still experienced lower than normal body mass. Lower BMI and slower puberty may be related to the number and severity of SCD complications, rather than hemoglobin phenotype alone. More studies are needed to better understand the effects of SCD severity and diet on somatic growth and puberty. Disclosures: No relevant conflicts of interest to declare.

Abstract 4852 Sickle Cell Disease (SCD) is a complex disease with various complications such as stroke, vaso-occlusive crisis (VOC), acute chest syndrome and leg ulcers. Sickle cell anemia (SCA; homozygous hemoglobin SS) is the most common form of SCD. Genetic variations and/or environmental modifiers may modulate clinical presentation of SCD. Few studies have examined hemoglobinopathies in Tunisia, North Africa. However, recently, frequencies of beta-thalassemia and sickle cell trait were estimated at 2.21% and 1.89%, respectively (Fattoum, 2006). In order to identify genetic factors that may predispose patients to SCD complications in this population, a pilot case control study was designed to assess polymorphisms in Human Platelet Antigen (HPA) Genes. HPA polymorphisms were recently associated with severe coronary artery disease in the general population in Tunisia (Abboud et al, 2010) and VOC presentation in SCA patients from Bahrain (Al-Subaie et al, 2009). We present here a study conducted in collaboration with the Department of Clinical Hematology at the Aziza Othmana Hospital in Tunis (Tunisia). The National Medical Ethics committee of Tunisia as well as the Institutional Review Board (IRB) of Morehouse School of Medicine (MSM) approved the study. Blood samples, clinical history and DNA samples were collected from SCD adult patients and healthy controls after informed consent. Previously validated questionnaires for genetic risks in patients with SCD (courtesy of Dr Telen, Duke University) were adapted to French. The Helena test kit was used to generate hemoglobin variant data in conjunction with cellulose acetate electrophoresis. Blood samples were collected in EDTA vacutainer tubes and genomic DNA was isolated,stored at −80°C and then shipped to MSM. Single nucleotide polymorphisms, SNPs (Table 1) were genotyped using PCR-RFLPs and compared with different clinical sub-phenotypes such as, onset age, strokes, cardiac problems, splenectomies, etc. as defined in the questionnaire. Pearson Chi-Square was used for comparison and a PCLeu33ProMspIHPA2rs606517GP1BA (2811)524 C>TMet145ThrSfaNIHPA3rs591117ITGA2B (3674)2622 T>GIle843SerFokITable 2:Tunisian cohort collection took place from January to December 2009. This table summarizes the number and genotypes of patients enrolled and questionnaires collectedSexMaleFemaleTotal (%)TOTAL455196 (100)SCD Patients403575 (78)Healthy Controls51621 (22)Hemoglobin Genotype of SCD patientsSS141529 (38.6)Sβ010919 (25.3)Sβ+112 (2.6)SC112 (2.6)SO101 (1.3)unknown13922 (29.3)Total (%)403575 (100)Questionnaires filled282654No questionnaires111021 Disclosures: No relevant conflicts of interest to declare.

Abstract 516 Children with sickle cell anemia (SCA) are at increased risk for stroke due to abnormal cerebral blood flow (CBF). The Stroke Prevention (STOP I) study showed that abnormal CBF could be detected using Trans-cranial Doppler (TCD), and may predict the risk for stroke. High TCD may be an indicator of main or branch cerebral artery vascular dysfunction, stenosis or fibrosis. Abnormal CBF is associated with cerebral ischemia and ultimately stroke. Brain derived neurotropic factor (BDNF) is a nerve growth factor with anti-apoptotic and neuro-protective properties. It is released in the central nervous system in response to ischemia. Platelet derived growth factor (PDGF) is an endothelial and smooth muscle mitogen. We hypothesized that circulating BDNF and PDGF levels will be elevated and may be associated with abnormal CBF as indicated by high TCD velocity measurements. Three groups of children, i.e. 1) SCA with abnormal TCDs (SATCD) - abnormal TCD ≥200 cm/sec, 2) SCA with normal TCDs (SNTCD) -normal TCD ≤170 cm/sec and 3) healthy controls were investigated. Stored, baseline plasma samples from the Stroke Prevention (STOP I) study and from an ongoing study of nutritional effects on inflammation in SCA children (NUTSCD) were analyzed in duplicate, using a multiplex antibody immobilized bead assay. Plasma levels of BDNF, PDGF AA and AB/BB and 10 other pro- and anti-inflammatory cytokines were assessed. A total of 39 samples (11 SATCD, 21 SNTCD and 7 controls) were analyzed. Of the 11 SATCD, 8 were eventually randomized to the standard care (SC), while 3 were in the transfusion (Tx) arm of STOP I. Since some of the SCA patients subsequently developed stroke, the levels of BDNF and PDGF in those who did vs. did not develop stroke were compared. Except where not applicable, values are presented as mean±SD. Chi-square was used to access distribution, while means were compared using t-test and analysis of variance (ANOVA). A Receiver Operator Characteristic (ROC) curve was used to access the sensitivity of BDNF and PDGF in predicting abnormal CBF as indicated by abnormal TCD measurement. The TCD velocity for SATCD group was 250±32 cm/s. There was no statistically significant difference in age or gender distribution among the groups. The mean body mass index was significantly lower for SCA subjects compared with controls (15±1kg/m2 vs. 23±7 kg/m2, p= 0.026). There was a statistically significant difference in mean BDNF levels, which was highest in SATCD compared with SNTCD (234±53 vs. 166±110 pg/ml, p=0.036) and controls (34±20 pg/ml, p High TCD velocity in children with SCA was associated with elevated BDNF and PDGF AA levels. Moreover, PDGF AA levels were significantly higher in the SCA patients who developed stroke. Further study is warranted to determine whether BDNF and/or PDFG AA, alone or with abnormal TCD, will better predict future stroke risk. Disclosures: No relevant conflicts of interest to declare.

More than half a century after the discovery of the molecular basis of Sickle Cell Disease (SCD), the causes of the phenotypic heterogeneity of the disease remain unclear. This heterogeneity manifests with different clinical outcomes such as stroke, vaso-occlusive episodes, acute chest syndrome, avascular necrosis, leg ulcers, priapism and retinopathy. These outcomes cannot be explained by the single mutation in the beta-globin gene alone but may be attributed to genetic modifiers and environmental effects. Recent advances in the post human genome sequence era have opened the door for the identification of novel genetic modifiers in SCD. Studies are showing that phenotypes of SCD seem to be modulated by polymorphisms in genes that are involved in inflammation, cell–cell interaction and modulators of oxidant injury and nitric oxide biology. The discovery of genes implicated in different phenotypes will help understanding of the physiopathology of the disease and aid in establishing targeted cures. However, caution is needed in asserting that genetic modifiers are the cause of all SCD phenotypes, because there are other factors such as genetic background of the population, environmental components, socio-economics and psychology that can play significant roles in the clinical heterogeneity.

The kinetics of urea metabolism were measured in children recovering from severe malnutrition. For a period of up to 10 d they received one of four diets which provided 711 kJ (170 kcal)/kg per d. Two groups received a diet with a high protein: energy (P:E) ratio of 10.6% (HP), enriched with either fat (HP/F) or maize starch and sucrose (HP/C). Two groups received a diet with a low P:E ratio of 8.8% (LP), enriched with either fat (LP/F) or maize starch and sucrose (LP/C). The rate of weight gain on the HP diets was significantly greater than on the LP diets. There was no difference in urea production between any of the four diets: HP/F 1.23 (SE 0.12), HP/C 1.37 (SE 0.14), LP/F 1.64 (SE 0.22), LP/C 1.15 (SE 0.15) mmol nitrogen/kg per h. On the HP diets urea excretion was 0.77 (SE 0.07) mmol N/kg per h. 61 % of production. There was significantly less urea excreted in the urine on diet LP/C than on LP/F (0.36 (SE 0.05) and 0.64 (SE 0.04) mmol N/kg per h respectively). A significantly greater percentage of the urea production was hydrolysed on the LP diets (61 %) compared with the HP diets (39 %), with the consequence that 50% of urea-N produced was available for synthetic activity on the LP diets compared with 30% on the HP diets. The increase in the urea hydrolysed on the LP diets was equivalent in magnitude to the decreased intake of N, so that overall intake plus hydrolysis did not differ between the LP and the HP diets. Crude N balance was similar on diets HP/F, HP/C and LP/C, but was significantly reduced on diet LP/F. These results show that there is an accommodation in urea kinetics during rapid catch-up weight gain, which becomes evident when the P:E ratio of the diet falls to 8.8%. It is proposed that, for a P:E ratio of 8.8%, protein is limiting for catch-up growth. When the intake has a P:E ratio of 8.8% the pattern of urea kinetics can be modified by the relative proportions of fat and carbohydrate in the diet. The measurement of urea kinetics provides a useful approach to the definition of the adequacy of the protein in the diet.

1. In rat studies, circulating concentrations of N-acetylneuraminic acid (NANA) have been shown to be an indicator of NANA concentrations in the brain and functional brain activity, in relation to nutritional state and stimulation. Abnormal behaviour can be improved with exogenous NANA. In the present study, the plasma NANA concentration has been measured in children with severe malnutrition and compared with that in controls.2. NANA was measured colorimetrically in the plasma of twenty-three severely malnourished children (mean age 11.43 (SD 6.05) months) before and after recovery, and in thirty-four controls (mean age 14.28 (SD 7.32) months). In thirteen of the malnourished children, NANA was measured after infections had been treated with a course of antibiotics.3. Mean plasma NANA concentration was significantly higher in protein-energy malnutrition (PEM)(2.89 (SD 0,58)μmol/ml; n 23) compared with controls (2.13(SD 0.37)μmol/ml; n 34,P< 0.001). The levels remained high in PEM after infections had been treated (2.87(SD 0.43) μmol/ml, n 13) but returned to control levels at recovery from PEM (2.14(SD 0.24)μmol/ml).4. In contrast to the findings in rats, in malnourished children plasma NANA concentrations were not reduced and did not relate directly to nutritional state or, by inference, brain function. These findings do not provide any support for the use of exogenous NANA supplements to improve brain function in humans.

1. The evidence is accumulating to suggest that glycine, the simplest amino acid, is conditionally essential in man. Benzoic acid, by conjugation with glycine to form hippuric acid, is known to deplete the free glycine pool of the body. Glycine is one substrate for the enzyme glutathione synthase (EC 6.3.2.3) and in the inborn error of metabolism in which glutathione synthase function is defective, increased quantities of 5-oxoproline are excreted in the urine.2. An oral dose of 4–10 g sodium benzoate was given to six normal adults to deplete the metabolic pool of glycine, and the urinary excretion of 5-oxoproline was followed for 6 h. In five of the six, a significant increase in the urinary 5-oxoproline was seen within 3 h.3. These-findings show that 5-oxoprolinuria can result from limited glycine availability, and may provide a useful test for assessing glycine sufficiency in a range of physiological and pathological states.