Plasma Citrate Concentration Research Articles

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Metabolomics analysis in adults with high bone mass identifies a relationship between bone resorption and circulating citrate which replicates in the general population

Objective Bone turnover, which regulates bone mass, may exert metabolic consequences, particularly on markers of glucose metabolism and adiposity. To better understand these relationships, we examined cross-sectional associations between bone turnover markers (BTMs) and metabolic traits in a population with high bone mass (HBM, BMD Z-score >=+3.2). Design beta-C-terminal telopeptide of type-I collagen (beta-CTX), procollagen type-1 amino-terminal propeptide (P1NP) and osteocalcin were assessed by electrochemiluminescence immunoassays. Metabolic traits, including lipids and glycolysis-related metabolites, were measured using nuclear magnetic resonance spectroscopy. Associations of BTMs with metabolic traits were assessed using generalized estimating equation linear regression, accounting for within-family correlation, adjusting for potential confounders (age, sex, height, weight, menopause, bisphosphonate and oral glucocorticoid use). Results A total of 198 adults with HBM had complete data, mean [SD] age 61.6 [13.7] years; 77% were female. Of 23 summary metabolic traits, citrate was positively related to all BTMs: adjusted beta(beta-CTX) = 0.050 (95% CI 0.024, 0.076), P = 1.71 x 10(-4), beta(osteocalcin) = 6.54 x 10(-4) (1.87 x 10(-4), 0.001), P = .006 and beta(P1NP) = 2.40 x 10(-4) (6.49 x 10(-5), 4.14 x 10(-4)), P = .007 (beta = increase in citrate (mmol/L) per 1 mu g/L BTM increase). Inverse relationships of beta-CTX (beta = -0.276 [-0.434, -0.118], P = 6.03 x 10(-4)) and osteocalcin (-0.004 [-0.007, -0.001], P = .020) with triglycerides were also identified. We explored the generalizability of these associations in 3664 perimenopausal women (age 47.9 [4.4] years) from a UK family cohort. We confirmed a positive, albeit lower magnitude, association between beta-CTX and citrate (adjusted beta(women) = 0.020 [0.013, 0.026], P = 1.95 x 10(-9)) and an inverse association of similar magnitude between beta-CTX and triglycerides (beta = -0.354 [-0.471, -0.237], P = 3.03 x 10(-9)). Conclusions Bone resorption is positively related to circulating citrate and inversely related to triglycerides. Further studies are justified to determine whether plasma citrate or triglyceride concentrations are altered by factors known to modulate bone resorption, such as bisphosphonates.

Measurement of the Concentration of Citrate in Human Biofluids in Patients Undergoing Continuous Renal Replacement Therapy Using Regional Citrate Anticoagulation: Application of a Two-Step Enzymatic Assay

Background: Continuous renal replacement therapy (CRRT) with regional citrate anticoagulation (RCA) is now commonly used to treat acute kidney injury in critically ill patients. The concentration of citrate is not routinely measured, with citrate accumulation and/or toxicity primarily assessed using surrogate measures. Objectives: The aim of this study was to measure the concentration of citrate in plasma and ultrafiltrate in patients receiving CRRT with RCA using a modified commercial enzymatic assay. Methods: After meeting inclusion criteria, blood was sampled from 20 patients before, during, and after episodes of filtration. Using spectrophotometry, samples were tested for citrate concentration. Demographic and other clinical and biochemical data were also collected. Throughout, a 15 mmol/L solution of trisodium citrate was used as the prefilter anticoagulant. Results were analysed using STATA (v15.0) and presented as mean (SD), median (IQR), or simple proportion. Comparisons were made using either the Student t test or the Wilcoxon rank-sum test. Correlation was assessed using Pearson’s r. Results: Twenty patients (17 males) were enrolled in the study. Mean (SD) age was 63.7 years (9.9). Median (IQR) ICU length of stay was 281 h (199, 422) with 85% undergoing intermittent positive pressure ventilation. Median APACHE 3 score was 95 (87, 117) with an overall 30% mortality rate. Median filtration time was 85 h (46, 149). No difference was found between pre- and post-filtration plasma citrate concentrations (79 µmol/L [50] vs. 71 µmol/L [42], p = 0.65). Mean citrate concentration during filtration was 508 µmol/L (221) with a maximum of 1,070 µmol/L. This was significantly higher than the pre/post levels (p < 0.001). Conclusions: Plasma concentrations of citrate rose significantly during episodes of CRRT using RCA returning back to normal after cessation of treatment.

The membrane protein ANKH is crucial for bone mechanical performance by mediating cellular export of citrate and ATP.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.

Metabolomics analysis in adults with high bone mass identifies a relationship between bone resorption and circulating citrate which replicates in the general population.

ObjectiveBone turnover, which regulates bone mass, may exert metabolic consequences, particularly on markers of glucose metabolism and adiposity. To better understand these relationships, we examined cross‐sectional associations between bone turnover markers (BTMs) and metabolic traits in a population with high bone mass (HBM, BMD Z‐score ≥+3.2).Designβ‐C‐terminal telopeptide of type‐I collagen (β‐CTX), procollagen type‐1 amino‐terminal propeptide (P1NP) and osteocalcin were assessed by electrochemiluminescence immunoassays. Metabolic traits, including lipids and glycolysis‐related metabolites, were measured using nuclear magnetic resonance spectroscopy. Associations of BTMs with metabolic traits were assessed using generalized estimating equation linear regression, accounting for within‐family correlation, adjusting for potential confounders (age, sex, height, weight, menopause, bisphosphonate and oral glucocorticoid use).ResultsA total of 198 adults with HBM had complete data, mean [SD] age 61.6 [13.7] years; 77% were female. Of 23 summary metabolic traits, citrate was positively related to all BTMs: adjusted ββ‐CTX = 0.050 (95% CI 0.024, 0.076), P = 1.71 × 10−4, βosteocalcin = 6.54 × 10−4 (1.87 × 10−4, 0.001), P = .006 and βP1NP = 2.40 × 10−4 (6.49 × 10−5, 4.14 × 10−4), P = .007 (β = increase in citrate (mmol/L) per 1 µg/L BTM increase). Inverse relationships of β‐CTX (β = −0.276 [−0.434, −0.118], P = 6.03 × 10−4) and osteocalcin (−0.004 [−0.007, −0.001], P = .020) with triglycerides were also identified. We explored the generalizability of these associations in 3664 perimenopausal women (age 47.9 [4.4] years) from a UK family cohort. We confirmed a positive, albeit lower magnitude, association between β‐CTX and citrate (adjusted βwomen = 0.020 [0.013, 0.026], P = 1.95 × 10−9) and an inverse association of similar magnitude between β‐CTX and triglycerides (β = −0.354 [−0.471, −0.237], P = 3.03 × 10−9).ConclusionsBone resorption is positively related to circulating citrate and inversely related to triglycerides. Further studies are justified to determine whether plasma citrate or triglyceride concentrations are altered by factors known to modulate bone resorption, such as bisphosphonates.

Albumin-corrected total/ionized calcium ratio is not superior to total/ionized calcium ratio as an indicator of citrate accumulation.

Total/ionized calcium ratio has been used as an indirect index of increased plasma citrate concentration during citrate anticoagulation. However, they were obtained without any adjustment of total calcium for albumin. Herein, we investigated whether the albumin-corrected total/ionized calcium ratio performed better than total/ionized calcium ratio. 20 critically ill patients were treated with continuous venovenous hemofiltration (CVVH) using citrate anticoagulation. The systemic total, ionized calcium and albumin levels were dynamically measured, and correlations between different calcium parameters and plasma citrate levels were analyzed. The highest correlation was found between plasma citrate level and total/ionized calcium ratio at the third hour (R = 0.88, p<0.001). The albumin-corrected total/ionized calcium ratio was not superior to total/ionized calcium ratio. The total/ionized calcium ratio at the third hour after the start of citrate-CVVH is an early and simple indicator to reflect increased plasma citrate level. Adjustment of systemic total calcium for serum albumin is not necessary.

Plasma citrate concentration: a possible biomarker for glaucoma in children

ObjectivesThe main aim of the present study was to examine a possible role of plasma and urine citrate levels as glaucoma indicators in school-aged children with glaucoma diagnosis.Patients34 school-aged children...

Continuous renal replacement therapy: a potential source of calories in the critically ill ,

Background: Overfeeding can lead to multiple metabolic and clinical complications and has been associated with increased mortality in the critically ill. Continuous venovenous hemofiltration (CVVH) represents a potential source of calories that is poorly recognized and may contribute to overfeeding complications.Objective: We aimed to quantify the systemic caloric contribution of acid-citrate-dextrose regional anticoagulation and dextrose-containing replacement fluids in the CVVH circuit.Design: This was a prospective study in 10 critically ill adult patients who received CVVH from April 2014 to June 2014. Serial pre- and postfilter blood samples (n = 4 each) were drawn and analyzed for glucose and citrate concentrations on each of 2 consecutive days.Results: Participants included 5 men and 5 women with a mean ± SEM age of 61 ± 4 y (range: 42-84 y) and body mass index (in kg/m2) of 28 ± 2 (range: 18.3-36.2). There was generally good agreement between data on the 2 study days (CV: 7-11%). Mean ± SEM pre- and postfilter venous plasma glucose concentrations in the aggregate group were 152 ± 10 and 178 ± 9 mg/dL, respectively. Net glucose uptake from the CVVH circuit was 54 ± 5 mg/min and provided 295 ± 28 kcal/d. Prefilter plasma glucose concentrations were higher in patients with diabetes (n = 5) than in those without diabetes (168 ± 12 compared with 140 ± 14 mg/dL; P < 0.05); however, net glucose uptake was similar (46 ± 8 compared with 61 ± 6 mg/min; P = 0.15). Mean ± SEM pre- and postfilter venous plasma citrate concentrations were 1 ± 0.1 and 3.1 ± 0.2 mmol/L, respectively. Net citrate uptake from the CVVH circuit was 60 ± 2 mg/min and provided 218 ± 8 kcal/d.Conclusions: During CVVH there was a substantial net uptake of both glucose and citrate that delivered exogenous energy and provided ∼512 kcal/d. Failure to account for this source of calories in critically ill patients receiving nutrition on CVVH may result in overfeeding.

Plasma Citrate Homeostasis: How It Is Regulated; And Its Physiological and Clinical Implications. An Important, But Neglected, Relationship in Medicine

The homeostatic maintenance of a normal plasma citrate concentration is an important factor in humans and in animals; and is required for many normal physiological activities. Dysregulation of normal plasma citrate presents pathophysiological hypocitricemic or hypercitricemic conditions. This can lead to clinical consequences in many areas of medicine; such as impaired blood clotting, altered acid/base status, impaired neuromuscular/cardiac activities, hypocitraturia and stone formation, bone disorders with loss of bone strength and increased fractures, hypocitricemia of surgical stress. These important implications of citrate relationships have been largely ignored by the contemporary clinical and biomedical community; to the extent that it is not even described in most current textbooks and review papers. This review describes the physiological, endocrine, and metabolic relationships in the normal regulation and maintenance of plasma citrate; and presents some important clinical consequences of its dysfunctional maintenance. The importance of bone, kidney and liver activities in the maintenance of normal plasma citrate is described along with the citricemic roles of parathyroid hormone, calcitonin and vitamin D. These factors and relationships are presented as the contemporary understanding of the integrated regulation of plasma citrate as the basis for its clinical importance in medicine. The exclusion of these citrate relationships leads to misunderstanding and misrepresentation of physiological and clinical conditions in many issues in medicine and paramedicine areas. The intent of this review is to revive the interest and support for research to address the many unknown and speculative issues of plasma citrate regulation and its important clinical implications. This is in the best interest of the medical community and the public-at-large.

Citrate pharmacokinetics at high levels of circuit citratemia during coupled plasma filtration adsorption.

The heparin requirement for coupled plasma filtration adsorption (CPFA) is usually high. Heparin administration often cannot be adherent to prescription, leading to a premature clotting of circuit and an insufficient volume of treated plasma. Regional citrate anticoagulation (RCA) could be an attractive alternative; however, no data are available on citrate pharmacokinetics at high levels of circuit citratemia. Fifteen septic shock patients with acute kidney injury undergoing CPFA with RCA at target circuit citratemia of 6 mmol/L were treated with CPFA-haemofiltration in pure predilution (CPFA-HF predilution group, n = 5 patients), or predilution haemodiafiltration (CPFA-HDF predilution group, n = 5 patients) or pre- and postdilution haemofiltration (CPFA-HF pre/postdilution group, n = 5 patients). Citrate pharmacokinetics was carried out through its determination in systemic and circuit blood, and effluent at time 0, 0.2, 1, 3, 6 and 9 h. The systemic concentrations of citrate in the CPFA-HF predilution group significantly increased over the sessions (from basal level of 0.21 to 0.76 mmol/L at 3 h), whereas they did not change in CPFA-HDF predilution and CPFA-HF pre/postdilution groups. Circuit plasma citrate concentrations (from 3 to 8 mmol/L) correlated strongly with circuit iCa++ levels (Spearman R = -0.7022, P < 0.01). Sieving coefficients of citrate were near the unit in all three groups and unrelated to blood and infusion flow rates in predilution. However, the amount of citrate removed by effluent was ∼40% for the CPFA-HF predilution group and reached 60% for both the CPFA-HDF predilution and CPFA-HF pre/postdilution groups (P < 0.05). As for the efficiency of plasmafiltration, the plasmafiltrate volume (from 17 to 20 mL/kg/day) was not significantly different among the groups. These results demonstrated that in refractory septic shock patients on CPFA at circuit citratemia of 6 mmol/L both HDF predilution and HF pre/postdilution were the best dialysis modalities to maintain a normal systemic citratemia through a high rate of citrate loss in the effluent.

Citrate Transport in NaDC1 Knockout Mice

Urinary citrate is the major endogenous inhibitor of calcium nephrolithiasis. Regulation of urinary citrate has been presumed to occur solely by the sodium dependent dicarboxylate cotransporter (NaDC1) located on the apical membrane of the proximal tubule. Using NaDC1 knockout (KO) and heterozygous (Het) mice (originally generated by Ho et al Kidney Int 2007) along with their wild type litter (WT) mates, we studied citrate and other Krebs cycle intermediates in plasma and urine with mice on either a normal diet (Nl) or on a 72 hour acid loading protocol (AL). Plasma citrate concentrations did not differ substantially in any of the conditions. On Nl diets, fractional excretion (FE) of citrate was higher in KO animals (0.24±0.02) than in WT (0.13±0.05); Hets were intermediate (0.18±0.04). Therefore, although KO of NaDC1 increases citrate excretion, substantial reabsorption of citrate still occurs in these animals (fractional reabsorption = 1-FE). With acidosis FE in WT was nearly abolished (0.003±0.001) but did not change in the KO animals (0.24± 0.02 Nl vs 0.25±0.0.02 AL diet). Surprisingly Het animals responded somewhat to acidosis but not completely, FE of 0.10±0.06 with AL and 0.18±0.04 with Nl diet. Urinary excretion of other dicarboxylates paralleled that of citrate except that urinary excretion of each decreased significantly in KO animals with acidosis. Thus, in examining FE in NaDC1 KO mice we found that most of renal reabsorption of citrate is not via NaDC1; although NaDC1 appears to account for the response to acidosis for citrate. Interestingly, the reabsorption of other Krebs cycle intermediates did respond significantly to acidosis. Also and surprisingly, Het animals do not have normal citrate reabsorption indicating that two normal alleles are important for normal citrate transport. NIH/NIDDK

Evidence that Osteoblasts are Specialized Citrate-producing Cells that Provide the Citrate for Incorporation into the Structure of Bone.

Citrate is a major component of bone in all vertebrates, but its implications in bone have remained largely unknown. Recent studies identified that citrate is incorporated into the structure of the hydroxyapatite nanocrystal/collagen complex; and is essential for the important biomechanical properties of bone. This raises the important question, “What is the source of citrate for incorporation into bone?”; A question that heretofore had remained unresolved. Studies in this report were designed to determine the plausibility of our concept that the osteoblasts are specialized citrate-producing cells, which provide the citrate that is incorporated into the structure of bone; and that osteogenic differentiation of mesenchyme cells leads to the development of the citrate-producing osteoblasts. The results demonstrated that primary human osteoblasts exhibit the capability of citrate-production. Undifferentiated mesenchyme cells do not exhibit the capability of citrate production; and osteogenic differentiation results in citrate-producing osteoblasts. The up-regulation of zinc uptake transporter ZIP1 is essential for the manifestation of the citrate-producing capability of the osteoblasts. We determined that osteoblast transport of citrate from plasma is not a likely source of citrate in bone. Thus, this study establishes for the first time that the osteoblasts are specialized citrate-producing cells that provide the citrate for incorporation into the structure of bone; and that mesenchyme cell osteogenesis leads to differentiated citrate-producing osteoblasts. This is a new understanding; which must include the osteogenic development of citrate-producing osteoblasts, and the process of “citration” in concert with mineralization during bone formation. It also provides a new understanding of the role of bone in the homeostatic maintenance of plasma citrate concentration.

Pharmacodynamic effects of EV-077: results of an in vitro pilot investigation in healthy volunteers

Antiplatelet agents are important for the treatment of patients with atherosclerotic disease manifestations. Antiplatelet agents act by inhibiting platelet activation and aggregation processes, which are mediated by several activating factors including thromboxane A2 (TxA2), adenosine diphosphate (ADP), and thrombin. In particular, TxA2 induced platelet activation is a pivotal pharmacological target as supported by the undisputable benefit of aspirin in secondary prevention of ischemic events [1, 2]. TxA2 is a metabolite of arachidonic acid (AA) deriving from the cyclooxygenase (COX)-1 enzyme, which transforms AA into prostaglandin H2 (PGH2), which is ultimately metabolized into TxA2 by thromboxane synthase (TS) [3]. Low dose of aspirin mainly inhibits COX-1 and subsequently TxA2 production. However, up to one-third of aspirin treated patients aspirin show persistent TxA2 production, which in turn exposes these subjects to an increased risk of atherothrombotic events [4]. These findings underscore the need to identify new TxA2 modulators. TxA2 receptor (TP receptor) antagonists, TS inhibitors, and dual TS inhibitors and TP receptor antagonists have been recently developed [3, 5]. Such antagonists and inhibitors have also beneficial effects on TP receptor mediated vascular inflammation and oxidative stress, thus on the progression and development of atherosclerotic lesions [6]. EV-077 is a novel dual TS inhibitor and TP receptor antagonist in early phases of clinical investigation. Limited data is currently available on the pharmacodynamic effects of this agent on human platelets [7]. In this pilot investigation we describe the pharmacodynamics effects on human platelets of escalating in vitro concentrations of EV-077. Ten healthy volunteers aged 35.1 ± 3.6 years old were recruited for this study. None of the subjects were taking any type of medication, including over the counter and herbal supplements. Venous blood was collected by antecubital puncture, in hirudin (25 lg/mL) and sodium citrate (0.150 M) containing tubes. Plasma citrate concentration was adjusted according to the haematocrit and the resulting free plasma citrate were adjusted to a final plasma concentration of 20 mmol/L as previously described [8]. Whole-blood hirudin-tubes were incubated with escalating concentrations of EV-077 (vehicle, 1, 10, 30, 100, and 300 nM) for 10 min at 37 8C. EV-077 was provided by Evolva SA (Reinach, Switzerland). Platelet-rich plasma (PRP) was obtained by centrifugation of whole-blood citrated tubes at 1179 g for 10 min. After measuring the platelet count, PRP was adjusted to 250,000/lL ± 10 %. This PRP was incubated with vehicle or 100 nM of EV-077 for 10 min at 37 C. Platelet aggregation was determined by impedance aggregometry using Multiplate Analyzer (MEA) (Dynabyte Medical, Munich, Germany) for whole-blood samples stimulated with AA (1 mM) (Dynabite Medical, Munich, Germany) and the TxA2 analog U-46619 (7 lM) (Calbiochem, Merck KGaA, Darmstadt, Germany), as well as with an agonist indirectly related to the TxA2 pathway, collagen (3 lg/mL) (Dynabite Medical, Munich, Germany). Lighttransmission platelet aggregometry (LTA) was performed using PRP by the turbidimetric method in a two-channel aggregometer (Chrono-Log 490 Model, Chrono-Log Corp., A. Tello-Montoliu F. Rollini B. Desai G. Pasqualino R. Patel D. J. Angiolillo (&) University of Florida College of Medicine-Jacksonville, 655 West 8th Street, Jacksonville, FL 32209, USA e-mail: dominick.angiolillo@jax.ufl.edu

Plasma Citrate Levels as a Potential Biomarker for Glaucoma

To determine the possibility of plasma citrate as a biomarker in patients with glaucoma. Twenty-one consecutive Caucasian patients with glaucoma and 21 sex- and age-matched controls were investigated. Plasma citrate, plasma creatinine, urine citrate, and urine creatinine were analyzed by ion chromatography. Mean (±standard deviation) concentrations and the calculated fractional citrate excretions were compared using the Mann-Whitney test. Sensitivity and specificity to detect glaucoma using plasma citrate levels were calculated. The mean plasma citrate (104.8±23.2 vs. 128.2±31.1 μmol/L; P=0.01) concentrations were significantly lower among the patients with glaucoma, whereas the mean urine citrate concentrations (1.7±0.9 vs. 2.8±1.9 μmol/L; P=0.07) were slightly lower. Mean plasma and mean urine creatinine concentrations showed no significant differences (plasma creatinine: 63.0±16.7 vs. 63.4±15.5 μmol/L; P=0.72; urine creatinine: 9.6±5.1 vs. 11.5±8.4 μmol/L; P=0.67). The calculated fractional citrate excretions were also not different with 12.1% versus 13.6% (P=0.37). Setting the cut-off limit at 110 μmol/L, the plasma citrate level evaluation would have a sensitivity of 66.7% and a specificity of 71.4% to detect glaucoma. In this masked study, plasma citrate levels were significantly decreased in Caucasian patients with glaucoma giving the possibility to use them eventually as a biomarker. The kidney function was normal in both groups, leaving the etiology of this hypocitraemia yet unexplained.

Evaluation of Quality Parameters for Cord Blood Donations

Background: Umbilical cord blood (CB) is widely used for hematopoietic stem cell transplantation and holds promise for the development of innovative medicinal products. In order to find out whether the conditions for collection and storage before processing might have an impact on the quality of CB preparations, viability and the clonogenic potential were assessed. Methods: CB was collected under field conditions. Flow cytometry was used to determine leukocytes, CD34/CD45+ cells, viability, and nucleated red blood cells (NRBC). Clonogenic activity was determined using isolated mononuclear cells (MNC). Results: Neither plasma citrate concentrations nor storage temperature (within 24 h) affected cell viability or colony formation. After storage for 49–80 h, leukocyte viability declined by about 16% compared to CB stored up to 24 h. In contrast, the clonogenic activity and CD34/CD45+ cell content were not affected. A higher gestational age was associated with a lower yield of clonogenic activity compared to midterm deliveries. NRBC varied widely (median 7.3%; range 0.63–17.3%) without relation to gestational age or colony formation. There was a close correlation between the percentage of viable CD34/CD45+ cells and colony formation (r = 0.77 for CFU-GM; r = 0.75 for CFU-C). Conclusions: The content of viable CD34/CD45+ cells represents the clonogenic activity of CB preparations. Therefore, determination of viable CD34/CD45+ cells should be generally performed as a routine quality control assay.

Enhanced standardization of the International Normalized Ratio through the use of plasma calibrants: a concise review.

The INR was introduced in 1983 as a means of standardizing the prothrombin time test and improving management of patients receiving warfarin-based oral anticoagulant therapy. Despite the eventual widespread use of the INR system, unacceptable levels of inter-laboratory variation persist. The use of certified plasma calibrants has been studied as a means to reducing INR variation. This review examines the pre-analytical and analytical variables contributing to INR problems. Also, the findings of several multicenter studies in which plasma calibrants were deployed are presented. Issues such as the number of calibrants used and their composition, calibrant format, plasma citrate concentration, reference thromboplastins used, methods of calibrant certification and alternative techniques for local system calibration are examined. The development of consensus guidelines on the use of plasma calibrants for INR standardization is recommended.

Protein C activity and antigen levels in childhood

Hereditary protein C deficiency is an important risk factor for thrombosis. To enable its diagnosis shortly after birth, we determined reference values of protein C antigen and activity levels for the first 3 months of life. To establish an age-related range of protein C levels we also determined median values for individuals up to 18 years of age. A good correlation between the two levels was seen from the 3rd/4th month of life onwards, whereas in the first 2 months the activity levels were significantly lower than the antigen levels. This was not due to interference by the increased plasma citrate concentration at high haematocrit values, and may suggest a dysfunctional protein C molecule in the neonatal period. We found a rapid rise in protein C activity and antigen levels until the age of 7-9 months, followed by a slower progression toward adolescence. In contrast to previous reports, our results indicate that adult values are probably not achieved until sometime during the 2nd decade of life.

Defective gallium-transferrin binding in Alzheimer disease and Down syndrome: possible mechanism for accumulation of aluminium in brain

The plasma distribution of gallium (as an analogue of aluminium) was investigated in patients with Alzheimer disease, Down syndrome, or stroke dementia, in subjects on haemodialysis for chronic renal failure, and in healthy controls. Gallium-transferrin binding was significantly lower in the Alzheimer (mean [SEM] 7·9 [1·1]%) and Down syndrome groups (6·9 [0·7]%) than in the controls (17·1 [1·6]%), whereas stroke dementia and haemodialysis patients had normal binding. There were no differences among the groups in plasma citrate concentration. The plasma transferrin concentration was slightly lower in the Alzheimer and Down syndrome groups than in the controls, but even lower in stroke dementia patients (1·74 [0·14] g/l vs 2·98 [0·18] g/l in controls). Transferrin iron saturation was higher in the Alzheimer (58·9%) and Down syndrome groups (81 ·6%) than in the controls (39·0%) or stroke dementia patients (33·4%). This deficiency of gallium/aluminium binding would leave more unbound aluminium which could move readily into the brain, where it has neurotoxic effects.

Altered plasma concentrations of glutamate, alanine and citrate in the early phase of acute myocardial infarction in man.

Plasma levels of glutamate, alanine, free fatty acids (FFA), citrate, glucose, insulin, lactate, creatine kinase and aspartate aminotransferase were determined frequently during the first 2-48 h after onset of chest pain in 10 patients who developed acute myocardial infarction (AMI) and in 8 who did not (non-AMI). An initial decrease in plasma glutamate and increase in alanine was found in AMI compared to non-AMI patients. The AMI group showed early, moderate rises of plasma FFA and citrate concentrations, positively related to the initial ST-segment elevation and to the enzymatic estimated infarct size. The AMI patients were continuously hyperglycaemic, but their relative insulin response i.e. plasma glucose/insulin ratio was identical to that of non-AMI patients. Lactate values did not differ between the two groups. Via participation in the malate-aspartate shuttle and by shunting pyruvate to alanine instead of lactate, glutamate is of importance for maintaining myocardial glucose utilization. Our finding of initial low plasma glutamate concentrations after onset of myocardial infarction suggests insufficient glutamate supply to the ischaemic myocardium. On basis of this and animal experiments, an external supply of glutamate might be a 'metabolic' treatment of AMI, alternative or additional to glucose-insulin-potassium infusion in order to promote myocardial glucose oxidation.

Pharmacokinetics of Heparin V: In Vivo and In Vitro Factors Affecting the Relationship Between Concentration and Anticoagulant Effect of Heparin in Rat Plasma

□ There are appreciable interindividual variations in rats of baseline activated partial thromboplastin time (APTT) and of the anticoagulant effect of heparin added to plasma (as reflected by the slope of the regression line describing the essentially linear relationship between In APTT and heparin concentration). Determination of baseline APTT and slope value on two occasions, 7 days apart, in the same rats revealed that (unlike in humans) these characteristics were subject also to considerable intraindividual variation. To explore the possible reasons for the observed variability, the effect of citrate concentration (acid citrate solution is used as a blood anticoagulant in the collection of plasma), calcium concentration (in the recalcifying solution used to initiate coagulation), and plasma incubation time (for activating the coagulation system) was determined. All three variables had pronounced effects on the anticoagulant response to heparin. Since rat erythrocytes are almost totally impermeable to citrate, hematocrit is a determinant of plasma citrate concentration when acid citrate solution is added in constant proportion to rat blood. Accordingly, inter- and intraindividual differences in baseline APTT and slope values were measured in another experiment in which the citrate solution to plasma (rather than blood) volume ratio was held constant and blood samples were obtained 30 days apart to permit the return of hematocrit values to normal. Intraindividual variation of the coagulation characteristics was appreciably decreased under these conditions. There are important differences between rats and humans with respect to the effect of citrate concentration and plasma incubation time on baseline APTT and on the anticoagulant action of heparin, as well as with respect to the relationship between these two characteristics.