Francisco V. Álvarez Menéndez

Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR

BACKGROUND: Recently cell-free plasma DNA has been described as a marker of apoptosis during hemodialysis (HD), but little is known about how different dialysis membranes may contribute to this process or whether pre-HD levels are restored afterwards. Here we evaluate the influence of the dialysis membrane on cell-free plasma DNA levels and investigate the clearance of plasma circulating DNA after HD. METHODS: Cell-free plasma DNA was measured using a real-time quantitative PCR for the beta-globin gene. Reference values for plasma DNA were established in a group of 100 healthy voluntary blood donors. Pre- and post-HD levels were also measured in 30 patients with end-stage renal disease on regular HD (52 sessions; 104 samples). The sessions lasted for 2.5-5 h. Different dialysis membranes were compared: high-flux (n=37) vs. low-flux (n=15) and polysulfone (n=42) vs. modified cellulose (n=10). To determine the time at which pre-HD levels are restored, DNA was quantified in serial plasma samples obtained from 10 of these 30 patients, just before and immediately after HD, as well as at 30, 60 and 120 min after HD. RESULTS: Reference plasma DNA values for healthy blood donors ranged from 112 to 2452 gEq/mL (median 740 gEq/mL). Cell-free plasma DNA levels significantly increased during HD (Wilcoxon test for paired samples, p<0.0001), with increases of more than four-fold observed in 75% of the patients after HD. There was no significant linear association between the length of the HD session (between 2.5 and 5 h) and the increase in cell-free plasma DNA concentration (Pearson correlation). No significant differences were observed between different types of membranes (Mann-Whitney U-test). Plasma DNA returned to pre-HD levels by 30 min after HD, regardless of the starting concentration. CONCLUSIONS: Plasma DNA levels significantly increase after a conventional 2.5-5-h HD session. Therefore, HD patients require special consideration for correct interpretation of plasma DNA concentrations. This parameter can be considered a reliable diagnostic tool for certain pathologies when measured at least 30 min after a HD session without further complications. The different dialysis membranes used in this study had no influence on cell-free plasma DNA concentrations, so the level of circulating DNA is not an appropriate marker of dialysis membrane biocompatibility.BACKGROUND Recently cell-free plasma DNA has been described as a marker of apoptosis during hemodialysis (HD), but little is known about how different dialysis membranes may contribute to this process or whether pre-HD levels are restored afterwards. Here we evaluate the influence of the dialysis membrane on cell-free plasma DNA levels and investigate the clearance of plasma circulating DNA after HD. METHODS Cell-free plasma DNA was measured using a real-time quantitative PCR for the beta-globin gene. Reference values for plasma DNA were established in a group of 100 healthy voluntary blood donors. Pre- and post-HD levels were also measured in 30 patients with end-stage renal disease on regular HD (52 sessions; 104 samples). The sessions lasted for 2.5-5 h. Different dialysis membranes were compared: high-flux (n=37) vs. low-flux (n=15) and polysulfone (n=42) vs. modified cellulose (n=10). To determine the time at which pre-HD levels are restored, DNA was quantified in serial plasma samples obtained from 10 of these 30 patients, just before and immediately after HD, as well as at 30, 60 and 120 min after HD. RESULTS Reference plasma DNA values for healthy blood donors ranged from 112 to 2452 gEq/mL (median 740 gEq/mL). Cell-free plasma DNA levels significantly increased during HD (Wilcoxon test for paired samples, p<0.0001), with increases of more than four-fold observed in 75% of the patients after HD. There was no significant linear association between the length of the HD session (between 2.5 and 5 h) and the increase in cell-free plasma DNA concentration (Pearson correlation). No significant differences were observed between different types of membranes (Mann-Whitney U-test). Plasma DNA returned to pre-HD levels by 30 min after HD, regardless of the starting concentration. CONCLUSIONS Plasma DNA levels significantly increase after a conventional 2.5-5-h HD session. Therefore, HD patients require special consideration for correct interpretation of plasma DNA concentrations. This parameter can be considered a reliable diagnostic tool for certain pathologies when measured at least 30 min after a HD session without further complications. The different dialysis membranes used in this study had no influence on cell-free plasma DNA concentrations, so the level of circulating DNA is not an appropriate marker of dialysis membrane biocompatibility.

Elevated transrenal DNA (cell-free urine DNA) in patients with urinary tract infection compared to healthy controls.

OBJECTIVE To study whether levels of transrenal DNA (Tr-DNA) are influenced by the presence of urinary tract infections (UTI). METHODS Tr-DNA concentrations were measured in 124 donors and 55 patients with UTI. Two methods for DNA extraction were compared. RESULTS UTI patients showed higher concentrations than the donors (normal range: 0-147 GE/mL). QIAamp MiniElute Virus Spin Kit was more efficient than QIAamp DNA Blood Mini Kit. CONCLUSION Increased concentrations of Tr-DNA urine DNA are shown in presence of UTI.

Neurofilament medium polypeptide (NFM) protein concentration is increased in CSF and serum samples from patients with brain injury

Abstract Background: Brain injury is a medical emergency that needs to be diagnosed and treated promptly. Several proteins have been studied as biomarkers of this medical condition. The aims of this study were to: 1) evaluate the selectivity and precision of a commercial ELISA kit for neurofilament medium polypeptide (NFM) protein; and 2) evaluate the concentration in cerebrospinal fluid (CSF) and serum of healthy individuals and patients with brain damage. Methods: An ELISA from Elabscience was used. The selectivity was evaluated using size-exclusion chromatography and mass spectrometry. Intra- and inter-batch coefficients of variation (CV) were also studied. Fifty-one CSF samples from 36 age-matched patients with hemorrhagic stroke (HS) (n=30), ischemic stroke (IS) (n=11) and healthy individuals (n=10) were assayed. In addition, serum samples from healthy volunteers (n=47), 68 serum samples from seven patients with HS, 106 serum samples from 12 patients with traumatic brain injury (TBI) and 68 serum samples from 68 patients with mild traumatic brain injury (mTBI) were also analyzed. Results: NFM was identified in the chromatographic fraction with highest immunoreactivity. The intra- and inter-batch CVs were ≤10% and ≤13%, respectively. The CSF-NFM concentration in HS was significantly higher (p<0.0001) than in IS and controls. Serum NFM concentration ranged from 0.26 to 8.57 ng/mL in healthy individuals (median=2.29), from 0.97 to 42.4 ng/mL in HS (median=10.8) and from 3.48 to 45.4 ng/mL in TBI (median=14.7). Finally, 44% of patients with mTBI had increased NFM concentration, with significantly higher levels (p=0.01) in patients with polytrauma. Conclusions: To our knowledge this is the first study describing increased NFM levels in CSF and serum from patients with brain damage.

Selenium levels and Glutathione peroxidase activity in the plasma of patients with type II diabetes mellitus

Selenium, an essential trace element, is involved in the complex system of defense against oxidative stress through selenium-dependent glutathione peroxidases (GPx) and other selenoproteins. Because of its antioxidant properties, selenium or its selenospecies at appropriate levels could hinder oxidative stress and so development of diabetes. In this vein, quantitative speciation of selenium in human plasma samples from healthy and diabetic patients (controlled and non-controlled) was carried out by affinity chromatography (AF) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) and isotope dilution analysis (IDA). Similarly, it is well known that patients with diabetes who exhibit poor control of blood glucose show a decreased total antioxidant activity. Thus, we evaluated the enzymatic activity of GPx in diabetic and healthy individuals, using the Paglia and Valentine enzymatic method, observing a significant difference (p<0.05) between the three groups of assayed patients (healthy (n=24): 0.61±0.11U/ml, controlled diabetic (n=38): 0.40±0.12U/ml and non-controlled diabetic patients (n=40): 0.32±0.09U/ml). Our results show that hyperglycemia induces oxidative stress in diabetic patients compared with healthy controls. What is more, glycation of GPx experiments demonstrated that it is the degree of glycation of the selenoenzyme (another species of the Se protein) what actually modulates its eventual activity against ROS in type II diabetes mellitus patients.

Variation of trace element concentrations in patients undergoing hemodialysis in the north of Spain

Abstract Background: Trace elements are essential substances for the proper physiological and biochemical functioning of the organism. Hemodialysis patients are potentially at risk of deficiency or excess of these elements. The application of inductively coupled plasma mass spectrometry (ICP-MS) allows the simultaneous quantification of very small amounts of multiple trace elements. The aim was to measure the serum concentration of copper (Cu), zinc (Zn), selenium (Se), and nickel (Ni), and the whole blood concentration of arsenic (As), lead (Pb), and manganese (Mn), in patients undergoing hemodialysis as well as in controls. Methods: The study was carried out in 57 hemodialysis patients compared with 57 controls with normal renal function. Serum and whole blood samples from the dialysis group were collected before and after hemodialysis sessions and Cu, Zn, Se, Ni, As, Pb and Mn levels were determined using ICP-MS. Results: Hemodialysis patients showed significantly lower blood levels of Cu, Zn and Se than controls (p < 0.001) and higher concentrations of Ni, As and Pb (p < 0.0001). The levels of Mn were similar in both groups. After performing hemodialysis, Cu, Zn, Se and Ni concentrations were significantly higher than the pre-hemodialysis levels (p < 0.0001). However, the concentration of As decreased (p < 0.0001) and Pb and Mn levels were not significantly altered after the dialysis session. Conclusion: Hemodialysis patients are at increased risk of trace elements deficiency (especially for Zn and Se) or excess (Ni) in respect to healthy subjects. Monitoring of blood levels and supplementation of some trace elements may be indicated in patients undergoing hemodialysis.

Challenges for Worldwide Harmonization of Newborn Screening Programs

BACKGROUND Inherited metabolic disorders (IMDs) are caused by a defect in a metabolic pathway, leading to malfunctioning metabolism and/or the accumulation of toxic intermediate metabolites. To date, hundreds of IMDs have been identified. Many of these diseases are potentially fatal conditions that are not apparent at birth. Newborn screening (NBS) programs involve the clinical and laboratory examination of neonates who exhibit no health problems, with the aim of discovering those infants who are, in fact, suffering from a treatable condition. CONTENT In recent years, the introduction of tandem mass spectrometry has allowed the expansion of screening programs. However, this expansion has brought a high degree of heterogeneity in the IMDs tested among different NBS programs. An attempt to harmonize the metabolic conditions recommended to be screened has been carried out. Two uniform screening panels have been proposed in the US and European Union, by knowledgeable organizations. Here, we review current evidence-based processes to assess and expand NBS programs. We also discuss the IMDs that have recently been introduced in some screening programs, such as severe combined immunodeficiencies, lysosomal storage disorders, and adrenoleukodystrophy. SUMMARY NBS programs have been an established public health function for more than 50 years to efficiently and cost-effectively identify neonates with severe conditions. However, NBS is not yet optimal. This review is intended to elucidate the current degree of harmonization of NBS programs worldwide as well as to describe the major controversial points and discuss the multiple challenges that must be confronted in expanded NBS strategies.

Strontium and oxidative stress in normal pregnancy

BACKGROUND Pregnancy brings about metabolic and oxidative changes that involve various trace elements and oxidative stress. Strontium (Sr) is a trace element scarcely studied in this context, although it has been suggested that it may play a role in the pathophysiology of preeclampsia. The main aim of this study was to evaluate Sr concentrations and oxidative status in normal pregnancy. METHODS The study population included non-pregnant women (n=31), healthy pregnant women in the first (n=50), second (n=51) and third (n=53) trimesters of gestation, and women in postpartum period (n=31). Additionally, samples from another twenty pregnant women were obtained in the three trimesters. Strontium, copper, selenium and zinc were measured by inductively coupled plasma-mass spectrometry. Calcium (Ca), uric acid (UA), lipid peroxidation and total antioxidant activity (TAA) were measured by spectrophotometric assays. RESULTS Strontium remained unchanged until the third trimester of pregnancy, in which significantly higher levels were found (p=0.001). The other elements showed diverse trends during pregnancy. Uric acid levels were significantly different in all groups (p<0.001), increasing gradually as the pregnancy progresses. In serial samples, there was a statistically significant positive correlation between Sr and gestational week of sampling (r=0.31, p=0.01), UA (r=0.40, p=0.001) and lipid peroxidation/TAA ratio (r=0.38, p=0.0002). Additionally, Sr correlated negatively with TAA (r=-0.40, p=0.0001). CONCLUSION Strontium seems to play a physiological role in the oxidative status of the human organism. Further studies involving Sr and pathologies of pregnancy are warranted.

Diagnosis of infection in critical care.

Sepsis is the primary cause of death in the intensive care unit. The prevention of sepsis complications requires an early and accurate diagnosis as well as the appropriate mon itoring. A deep knowledge of the immunologic basis of sepsis is essential to better understand the scope of incorporating a new marker into clinical practice. Besides revising this theoretical aspect, the current available tools for bacterial iden tification have been briefly reviewed as well as a variety of new markers showing either well-recognized or potential usefulness for diagnosis and prognosis of infections in crit ically ill patients. Particular conditions such as community acquired pneumonia, pedi atric sepsis, or liver transplantation, among others, have been separately treated, since the optimal approaches and markers might be different in these special cases.

Strontium and its role in preeclampsia

BACKGROUND Preeclampsia (PE) is considered a specific vascular disease in which endothelial dysfunction may be the crucial factor of its pathogenesis. It has been suggested that strontium (Sr) may play a role in the pathophysiology of PE. Our group established in a previous study the serum levels of Sr in healthy pregnancies, and the main aim of the present study was to evaluate Sr concentrations and oxidative status in preeclamptic women. METHODS The study population included women with early-onset PE (E-PE, n = 39), late-onset PE (L-PE, n = 67) and serial samples from a subset of preeclamptic women (PE-ss, n = 20). The control group included women with gestational hypertension (GH, n = 56) and healthy pregnancies (samples collected in the 1st (n = 50), 2nd (n = 51) and 3rd trimesters (n = 53)). Strontium, calcium (Ca), uric acid (UA), placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFlt-1), N-terminal pro-brain natriuretic peptide (NT-proBNP), lipid peroxidation and total antioxidant activity (TAA) were measured in these samples. RESULTS Mean Sr levels were significantly higher in PE than in control groups (p ≤ 0.0001). Calcium values were found to be significantly lower in E-PE compared to control groups (p = 0.03). Higher levels of NT-proBNP were found in PE vs. control groups (p < 0.001). sFlt-1/PlGF ratio was higher in E-PE compared to L-PE and GH (p < 0.001). Uric acid levels in PE were significantly higher than in control groups (p < 0.0001). There was a strong positive correlation between UA and Sr in the E-PE serial samples (r = 0.80, p < 0.0001). Lipid peroxidation and lipid peroxidation/TAA ratios were found to be higher in PE, with lower values of TAA. CONCLUSION The higher levels of Sr and the alterations of redox status found in preeclamptic women, along with the strong correlation between UA and Sr suggest that this element may be involved in the pathogenesis of PE.