The role of urinary N-acetyl-ß-D-glucosaminidase in early detection of acute kidney injury among pediatric patients with neoplastic disorders in a retrospective study.

BACKGROUND: The 1-year cumulative incidence of AKI reportedly is high (52%) in pediatric neoplastic disorders. About half of these events occur within 2 weeks. However, subclinical AKI episodes may remain unrecognized by the conventional creatinine-based approaches. We investigated the diagnostic value of urinary N-acetyl-ß-D-glucosaminidase (uNAG) as an early marker of acute kidney injury (AKI). METHODS: In our retrospective study, 33 children with neoplastic disorders were inculded who had serial uNAG tests (at least 5 samples/patient) with a total of 367 uNAG measurements. Renal function was determined by cystatin-C and creatinine based GFR, and relative increase of uNAG index (uNAGRI). We focused on detecting both clinical and subclinical AKI episodes (according to Biomarker-Guided Risk Assessment using pRIFLE criteria and /or elevated uNAG levels) and the incidence of chronic kidney damage. RESULTS: Sixty episodes in 26 patients, with positivity at least in one parameter of kidney panel, were identified during the observation period. We detected 18/60 clinical and 12/60 subclinical renal episodes. In 27/60 episodes only uNAG values was elevated with no therapeutic consequence at presentation. Two patients were detected with decreased initial creatinine levels with 3 "silent" AKI. In 13 patients, modest elevation of uNAG persisted suggesting mild, reversible tubular damage, while chronic tubuloglomerular injury occurred in 5 patients. Based on ROC analysis for the occurence of AKI, uNAGRI significantly indicated the presence of AKI, the sensitivity and specificity are higher than the changes of GFRCreat. Serial uNAG measurements are recommended for  the reduction of the great amount of false positive uNAG results, often due to overhydratation. CONCLUSION: Use of Biomarker-guided Risk Assessment for AKI identified 1.5 × more clinical and subclinical AKI episodes than with creatinine alone in our pediatric cancer patients. Based on the ROC curve for the occurence of AKI, uNAGRI has relatively high sensitivity and specificity comparable to changes of GFRCysC. The advantage of serial uNAG measurements is to decrease the number of false positive results. TRIAL REGISTRATION: The consent to participate is not applicable because it was not reqired for ethical approval and it is a retrospectiv study.

Detection of haemolysis, a frequent preanalytical problem in the serum of newborns and adults.

BACKGROUND: Preanalytical problems can be more frequent in case of preterm and term newborns as compared to the general patient population. Here we present the leading preanalytical errors in our laboratory, the prevalence of haemolysis and its impact on laboratory test results, and our efforts to improve the diagnostic workup of newborns' samples. METHODS: Preanalytical quality indicators were analysed in all samples in 2018. The haemolysis index was measured spectrophotometrically in serum samples in the period of 2012-2018, and the ratio of haemolysed samples and the test rejection rates were analysed. The data of newborns and other patients were analysed separately. RESULTS: During the tested year, the leading preanalytical errors were haemolysis in serum samples, inadequate sample identification and clotting of anticoagulated blood regarding all samples or newborns. In this seven-year period the ratio of haemolysed serum samples was 4.00% in all patients and 46.4% in newborns, while the test rejection rates due to haemolysis were 0.57% and 3.71%, respectively. Haemolysis indices were significantly higher in case of newborns than in patients with documented severe intravascular haemolysis which suggests that the major reason of elevated haemolysis indices in newborns was in vitro haemolysis. Accordingly, all C-reactive protein (CRP) results which were rejected by severe haemolysis became reliable after repeating blood sampling. CONCLUSION: Haemolysis is the leading preanalytical problem not only in newborns but also in the general patient population. Our study highlights the importance of automated assessment of serum indices and continuous monitoring of the preanalytical quality indicators and suggests the need for education and blood collection trainings.

Intracardiac Fibrinolysis and Endothelium Activation Related to Atrial Fibrillation Ablation with Different Techniques.

OBJECTIVE: The effect of pulmonary vein isolation (PVI) on fibrinolytic and endothelial activation with currently applied periprocedural anticoagulation has not been explored. We measured markers of fibrinolysis and endothelium activation before and after PVI with the second-generation cryoballoon (Cryo), pulmonary vein ablation catheter (PVAC-Gold), and irrigated radiofrequency (IRF). METHODS: Markers of fibrinolysis and endothelium activation in left atrial (LA) blood samples were measured in 31 patients before and after PVI (Cryo:10, PVAC-Gold: 7, IRF: 14). Periprocedural anticoagulation included uninterrupted vitamin K antagonist and iv heparin (ACT≥300 sec) during LA dwelling. RESULTS: Levels of D-dimer (median; interquartile range, mgFEU/L) increased with all techniques (PVAC: 0.34; 0.24-0.50 versus 0.70; 0.61-1.31; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; PAP complex level (ng/ml) increased after Cryo (247.3, 199.9-331.6 versus 270.9, 227.9-346.7; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; PAI-1 activity (%) decreased with the PVAC (1.931; 0.508-3.859 versus 0.735, 0.240-2.707; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; VWF antigen levels and FVIII activity increased after PVI with all the 3 techniques. The levels of soluble VCAM-1 (ng/ml) did not change after PVAC procedures, but increased after Cryo (542, 6; 428.5-753.1 versus 619.2; 499.8-799.0; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93. CONCLUSION: PVI with contemporary ablation techniques and periprocedural antithrombotic treatment induces coagulation and endothelium activation of similar magnitude with different ablation methods.

Comparative erythrocyte deformability investigations by filtrometry, slit-flow and rotational ektacytometry in a long-term follow-up animal study on splenectomy and different spleen preserving operative techniques: Partial or subtotal spleen resection and spleen autotransplantation.

BACKGROUND: Partial or subtotal spleen resection or spleen autotransplantation can partly preserve/restore the splenic filtration function, as previous studies demonstrated. OBJECTIVE: For better evaluation and follow-up of the various spleen-preserving operative techniques' effectiveness versus splenectomy, a composite methodological approach was applied in a canine experimental model. METHODS: Beagle dogs were subjected to control (n = 6), splenectomy (SE, n = 4), partial and subtotal spleen resection (n = 4/each) or spleen autotransplantation groups (AU, Furka's spleen-chip method, n = 8). The follow-up period was 18 postoperative (p.o.) months. Erythrocyte deformability was determined in parallel by bulk filtrometry (Carat FT-1 filtrometer), slit-flow ektacytometry (RheoScan D-200) and rotational ektacytometry (LoRRca MaxSis Osmoscan). RESULTS: By filtrometry, relative cell transit time increased in the SE group (mostly in animal Nr. SE-3), showing the highest values on the 3rd, 9th and in 18th p.o. months. Elongation index values decreased in this group (both by slit-flow and rotational ektacytometers). In general, AU and two resection groups' values were lower versus control and higher than in SE. CONCLUSIONS: Forasmuch in the circulation both elongation by shear stress and filtration occur, these various erythrocyte deformability testing methods together may describe better the alterations. Considering the possible complications related to functional asplenic-hyposplenic conditions, individual analysis of cases is highly important.

Altered lipid subfraction profile and impaired antioxidant defense of high-density lipoprotein in Smith-Lemli-Opitz syndrome.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is a rare disease caused by biallelic mutation in the 7-dehydrocholesterol (7DHC) reductase gene. High oxidizability of 7DHC and the appearance of small-sized low-density lipoprotein (LDL) subfractions indicate increased endogenous oxidative stress that is counterbalanced by natural antioxidant defense mechanisms including the high-density lipoprotein (HDL)-associated paraoxonase-1 (PON1) enzyme. PON1 prevents lipoproteins from oxidative modifications; however, PON1 activity and the distribution of lipoprotein subfractions have not been studied in SLOS. METHODS: 7DHC levels and PON1 arylesterase activities were measured spectrophotometrically in 11 SLOS patients and 10 healthy children. Lipoprotein subfractions were detected by polyacrylamide gel electrophoresis. RESULTS: Compared to controls, there was a shift towards the small-dense LDL subfraction and the large HDL subfraction in SLOS. PON1 arylesterase activity was significantly decreased in SLOS patients and correlated negatively with the proportion of small-dense LDL subfraction and the proportion of large HDL subfraction. Significant positive correlations were detected between PON1 arylesterase activity and the ratios of intermediate and small HDL subfractions. CONCLUSIONS: Decreased PON1 activity and the deleterious shift in the distribution of lipoprotein subfractions may contribute to the impaired antioxidant status observed in SLOS. Monitoring of serum PON1 arylesterase activity may be a complementary biomarker in SLOS.

Rosuvastatin improves impaired endothelial function, lowers high sensitivity CRP, complement and immuncomplex production in patients with systemic sclerosis--a prospective case-series study.

INTRODUCTION: We studied the effect of rosuvastatin on endothelial and macrovascular function, cardiovascular risk factors and the complement pathway in patients with systemic sclerosis (SSc). METHODS: Altogether 28 patients with SSc underwent laboratory and complex vascular assessments before and after six months of 20 mg rosuvastatin treatment. Flow-mediated dilation (FMD) of the brachial artery, as well as carotid artery intima-media thickness (ccIMT), carotid-femoral and aorto-femoral pulse wave-velocity (PWV) were analyzed by ECG-synchronized ultrasound. Ankle-brachial index (ABI) was determined by Doppler, and forearm skin microcirculation was assessed by Laser Doppler perfusion monitoring. RESULTS: Brachial artery FMD significantly improved upon rosuvastatin therapy (2.2% ± 3.3% before versus 5.7% ± 3.9% after treatment, P = 0.0002). With regard to patient subsets, FMD significantly improved in the 21 lcSSc patients (from 2.1% to 5.6%, P = 0.001). In the seven dcSSc patients, we observed a tendency of improvement in FMD (from 3% to 6%, P = 0.25). Changes in PWV, ccIMT and ABI were not significant. Mean triglyceride (1.7 ± 0.97 versus 1.3 ± 0.46 mmol/l, P = 0.0004), total cholesterol (5.3 ± 1.6 mmol/l versus 4.2 ± 1.3 mmol/l, P = 0.0003), low density lipoprotein cholesterol (3.0 ± 1.3 versus 2.2 ± 1.0 mmol/l, P = 0.005) and C-reactive protein levels (CRP) (5.1 ± 5.2 versus 3.4 ± 2.7, P = 0.01) levels significantly decreased after rosuvastatin treatment. Mean C3, C4 and IC levels also decreased significantly as compared to pretreatment values. CONCLUSIONS: Six-month rosuvastatin therapy improves endothelial function and lowers CRP, C3, C4 and IC levels indicating possible favourable effects of this statin on the cardiovascular and immune system in SSc.

Relation between biomarkers and clinical severity in patients with Smith-Lemli-Opitz syndrome.

UNLABELLED: Smith-Lemli-Opitz syndrome (SLOS), a multiple congenital anomaly with severe mental retardation, is caused by decreased activity of 7-dehydrocholesterol reductase. Fifteen Hungarian patients were diagnosed with SLOS on the basis of clinical symptoms, serum cholesterol, 7-dehydrocholesterol, and molecular genetic testing. Their age at the time of diagnosis in mild SLOS (n = 4, clinical score <20) was 0.5-18 years, cholesterol was 2.37 ± 0.8 mmol/L, and 7DHC was 0.38 ± 0.14 mmol/L. In the group of typical SLOS (n = 7, score 20-50), the diagnosis was set up earlier (age of 0.1-7 years); t-cholesterol was 1.47 ± 0.7 mmol/L, and 7DHC was 0.53 ± 0.20 mmol/L. Patients with severe SLOS (n = 4, clinical score > 50) died as newborns and had the lowest t-cholesterol (0.66 ± 0.27 mmol/L), and 7DHC was 0.47 ± 0.14 mmol/L. Correlation coefficient with clinical severity was 0.74 for initial t-cholesterol and 0.669 for Cho/7DHC. Statistically significant difference was between the initial t-cholesterol of mild and severe SLOS (p = 0.01), and between the Cho/7DHC ratios of groups (p = 0.004). In severe SLOS, the percentage of α-lipoprotein was significantly lower than in typical (p = 0.003) and mild SLOS (p = 0.004). Although serum albumin, total bilirubin, and hemostasis parameters remained in the reference range during cholesterol supplementation (n = 10) combined with statin therapy (n = 9), increase of aspartate aminotransferase and alanine aminotransferase in 50 % of the patients probably refers to a reversible alteration of liver function; therefore, statin therapy was suspended. CONCLUSION: life expectancy is fundamentally determined by the initial t-cholesterol, but dehydrocholesterol and α-lipoprotein have prognostic value. Accumulation of hepatotoxic DHC may inhibit the synthesis of α-lipoproteins, decreasing the reverse cholesterol transport. During statin therapy, we suggest monitoring of lipid parameters and liver function.

Characterization of a novel high-density lipoprotein antioxidant capacity assay and its application to high-density lipoprotein fractions.

OBJECTIVES: High-density lipoprotein (HDL) inhibits low-density lipoprotein (LDL) oxidation therefore it is involved in the prevention of atherogenesis. HDL particles originating from different persons possess different antioxidant activities. Our aim was to establish a method for the measurement of HDL antioxidant capacity, which is suitable for testing the antioxidant activity of HDL samples in a wide range and produces data relevant to in vivo HDL-LDL interactions. Hemin was used as pro-oxidant since its role in the course of LDL oxidation and atherosclerosis is proven. METHODS: Hemin-induced and hydrogen peroxide catalyzed lipid peroxidation of LDL was performed in the presence and absence of HDL. The time interval required for reaching the maximum reaction velocity (ΔT(Vmax)) was determined and HDL antioxidant capacity was expressed as the ratio of the ΔT(Vmax) with and without HDL. HDL fractions (n=8) isolated by ultracentrifugation from healthy donors were analyzed and their antioxidant capacities were compared. RESULTS: In parallel with their increasing density, HDL fractions expressed increasing antioxidant capacity (106.12-194.12%). Within-run and within-laboratory CVs of the method were 1.72-1.87% and 4.09-4.93%, respectively. Alterations of hydrogen peroxide concentration in the range of 50-125 µmol/L did not influence the assay results, while the elevation of hemin concentration (between 3 and 9 µmol/L) resulted in decreased antioxidant capacity. The values for hemin degradation correlated well with conjugated diene formation. CONCLUSIONS: Hemin-induced LDL oxidation is a reliable assay system to test the antioxidant capacity of HDL and its subpopulations.

Monitoring of mycophenolic acid and kidney function during combined immunosuppressive therapy.

BACKGROUND: Mycophenolic acid (MPA), a selective inhibitor of lymphocyte proliferation, has lately been used to improve renal function and prolong graft survival in renal transplanted patients. Still, there is no consensus considering the recommended dosing and the therapeutic range of MPA. METHODS: To estimate the safe therapeutic range of MPA, its plasma level and indicators of kidney function were measured in 216 patients (138 male, 78 female, age 46 ± 12 years) 67 ± 46 months after transplantation. Besides MPA, patients received cyclosporine (Group A, n=122) or tacrolimus (Group B, n=77). Seventeen patients (Group C) were treated with MPA in combination with everolimus or sirolimus. Plasma MPA was measured by enzyme inhibition assay. RESULTS: In the whole study group MPA level increased with the dose of MPA (p=0.013). MPA level was below the therapeutic range in 40% (Group A) and 45% (Group B) of patients, respectively. MPA was 1.9 ± 1.56 mg/L in Group A, 2.4 ± 1.69 mg/L in Group B. In Group A MPA level increased and cyclosporine decreased with the progress of renal disease. CONCLUSIONS: Increasing MPA/cyclosporine ratio at more severe stages of chronic kidney disease was tolerable for the patients and rejection could be avoided. Tubular damage detected by urinary N-acetyl-ß-D-glucosaminidase did not correlate with the MPA level.

A patient with Smith-Lemli-Opitz syndrome: novel mutation of the DHCR7 gene and effects of therapy with simvastatin and cholesterol supplement.

BACKGROUND: The Smith-Lemli-Opitz (SLO) syndrome is a multiple congenital anomaly with mental retardation due to a decreased or lack of activity of 7-dehydrocholesterol reductase as a consequence of mutations of the DHCR7 gene. This paper describes a special patient with SLO syndrome. Laboratory examination showed low cholesterol (2.77 mmol/L) and increased 7-dehydrocholesterol level (102 mg/L). Molecular genetic analysis revealed a compound heterozygosity c.964-1G>C/p.G366V (c.G1370T) of the proband. The p.G366V is a novel mutation of the DHCR7 gene with guanine by thymine nucleotide exchange resulting in glycin by valin amino acid exchange in the dehydrocholesterol reductase enzyme. Simvastatin (0.2 mg/kg/day) and cholesterol replacement therapy (150-250 mg/kg/day) led to significant improvement in the patient's laboratory findings (7-dehydrocholesterol, cholesterol) as well as in his behavior and gross motor function. CONCLUSION: Our patient demonstrates that the c.964-1G>C/p.G366V (c.G1370T) genotype of combined heterozygosity is associated with a typical form of SLO syndrome along with moderately altered laboratory findings and a favorable biochemical response to cholesterol and simvastatin treatment.

Hepatobiliary response in postoperative lipid therapy in gastrointestinal surgery.

BACKGROUND/AIMS: Intravenous lipid emulsions may contribute to the development of total parenteral nutrition (TPN)--induced hepatobiliary complications. METHODS: In a prospective, randomised setting the authors compared the short-term hepatic effects of medium-chain triglycerides/short-chain triglycerides (MCT/LCT) physical mixture with a four-component intravenous (i.v.) lipid emulsion (LCT, MCT, Olive-oil and Fish-oil) in patients undergoing elective gastrointestial surgery during the early postoperative period. RESULTS: The authors demonstrated that total and conjugated bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate amino transferase and cholinesterase did not change significantly during the 5-days observation period. In contrast to this, gamma-glutamyl transferase (GGT) activity increased by 2,4 times during 5-days therapy with the lipid emulsions mentioned above (SMOF lipid: 21,9 to 52,9 U/L, Lipofundin: from 32,5 to 79,6 U/L). CONCLUSION: during a 4-days administration hepatic effect of the intravenous lipid emulsions did not differ significantly. The changes in enzyme levels confirm the cholestatic type of hepatobiliary deviations without clinical impact on short-term TPN therapy.

Identification of sulfhemoglobinemia after surgical polypectomy.

Sulfhemoglobinemia (SHb) is an uncommon cause of cyanosis that is predominantly drug-induced in adults. We report an unusual case of sodium sulfate-induced sulfhemoglobinemia in a 61-year-old woman after surgical polypectomy. Fractional hemoglobin derivates were assayed by spectrophotometry and high-performance liquid chromatography. The SHb ratio was 8.6% in the first sample and 3.77% a month later measured by spectrophotometry. In the blood hemolysate, a new peak was identified as SHb with high-performance liquid chromatography (HPLC). HPLC showed the presence of 9.37% SHb in the first sample and 4.88% a month later. After removing the suspected toxic agent the cyanosis decreased significantly. The findings underline the importance of routine SHb detection in cyanosis of unknown origin especially in emergency cases.

Late effects on renal glomerular and tubular function in childhood cancer survivors.

BACKGROUND: Late nephrotoxicity among childhood cancer survivors is poorly documented. METHODS: We investigated 115 patients and 86 controls assessing serum cystatin C concentration (CysC), urinary N-acetyl-beta-D-glucosaminidase activity (NAG), and microalbuminuria. Proteinuria was quantified and electrophoresis performed. Polymorphism of the angiotensin convertase enzyme (ACE) gene was determined by genomic PCR. RESULTS: CysC was elevated in Wilms tumor (WT) patients. Gross proteinuria was observed in 30 patients including three patients with progressive proteinuria who improved on ACE-inhibitor treatment. Neither patients with proteinuria nor the entire study population differed from controls with respect to ACE polymorphism. Pathologically elevated urinary NAG was noted in 38% of leukemia/lymphoma, 54% of solid tumor, 20% of WT survivors. A similar distribution of pathological microalbuminuria was found. CONCLUSIONS: Mild-to-moderate subclinical glomerular and tubular damage can be identified in many childhood cancer survivors. However, most patients experience some spontaneous recovery from acute nephrotoxicity.

Cystatin C is a suitable marker of glomerular function in children with cancer.

Antineoplastic chemotherapy is associated with nephrotoxic side effects. Data on nephrotoxicity in childhood cancer are scanty, in part because of the difficulties in obtaining reliable markers of glomerular function. We used serum cystatin C (cysC) to assess glomerular function. CysC was compared with serum creatinine concentration (S(Cr)), the endogenous creatinine clearence ( C(Cr)), and the calculated Counahan formula ( C(Counahan)) in children with leukemia and solid tumors. CysC was measured by particle-enhanced immunoturbidimetric assay. Serum and urinary creatinine concentrations were determined by the Jaffé method. Samples were obtained from 258 children, including 92 receiving anticancer chemotherapy, 108 long-term survivors, 40 children without any renal disease, and 18 patients with chronic renal insufficiency. CysC of patients on current chemotherapy was assessed both before and after treatment. Significant correlations were found between cysC and S(Cr) and between 1/cysC and C( Counahan). CysC increased significantly after cisplatin, methotrexate, cyclophosphamide, ifosfamide, and multimodality treatment. Our results suggest that cysC measurement can be used to characterize glomerular function in children with cancer.

Age dependence of serum beta-N-acetylhexosaminidase (NAG) activity.

Serum N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30) is a hexosaminidase and may be a predictor of vascular injury, e.g., in infant respiratory distress syndrome, pneumonia, broncho-pulmonary dysplasia and necrotizing enterocolitis. To estimate the new diagnostic prospects we have modified our urinary NAG assay. In this sensitive colorimetric micro-assay, VRA-GlcNAc was used as a substrate. In the present study the age dependence of serum NAG activity was investigated in newborn babies, infants (1-24 months), children (2-18 years) and adults (19-80 years). Serum NAG activity was found to be age-dependent; it is higher in early childhood (11-59 U/l) but decreases to a constant value at the age of 1-2 years. After the age of 2 years it is similar to adults' NAG (10-30 U/l). In pediatrics age-matched reference ranges must be taken into consideration.

Urinary homogentisic acid in alkaptonuric and healthy children.

To detect and follow-up the metabolic status of patients with alkaptonuria (AKU), urinary homogentisic acid (HGA) was measured by gas chromatography. These results were close to values we obtained by colorimetric method (linearity: upto 700 mg/l, detection limit: 1 mg/l, within-run imprecision (CV): 1.2% at 100 mg/l HGA, 4.9% at 10 mg/l, between-run CV: 6.8% at 100 mg/l). To determine urinary reference ranges of HGA, 84 healthy children (age: 2 months-18 years) were divided into five age groups. HGA and creatinine were measured in their morning urine. Statistical analysis proved that urinary HGA/creatinine ratio is age-dependent. The ratio is relatively high between 1 and 6 years of age, with large scatter (upper limit of reference ranges given as mean + 2 SD: 5.5-7.2 mg/mmol = 0.03-0.04 mmol/mmol creatinine), and it decreases with age. Approximately at the age of 7 years, HGA/creatinine ratio becomes constant, and later it is similar to the adult value (upper limit: 2.8 mg/ mmol = 0.017 mmol/mmol creatinine). We monitored a patient during her 1-5th year of life, and her urinary HGA was 80-200 times higher than the upper limit of the age-matched reference ranges. The measurement of HGA supports the decision for starting restricted protein diet and is useful for the evaluation of the effectiveness of therapy.