Validation of a reference method for total cholesterol measurement in human serum and assignation of reference values to proficiency testing samples

ObjectivesOur objective was to develop a reference method to measure total cholesterol in human serum, in order to assign values and assess the accuracy of field methods in French clinical laboratories.Design and methodsA reference method based on gas chromatography coupled with mass spectrometry and isotope dilution (GC–IDMS) was developed and validated. It was then used to assign reference values to five frozen serum samples from voluntary proficiency testing schemes gathering 170 French clinical laboratories. Three peer groups were defined and bias against the reference method target value was calculated.ResultsAccuracy of the reference method was assessed against NIST SRM 1951b. Bias of the reference method was less than 0.5% and imprecision was less than 1.0%. Our study indicated that field methods tended to overestimate total cholesterol concentration, mean bias being + 5.02% ± 1.02%. The most popular methods (phenolic chromogen with spectrophotometric detection, 80% of participants) exhibited the highest bias (peer group mean bias: + 5.51 ± 1.24%). Neither these methods nor those using a non-phenolic chromogen with reflectometric detection (10% of participants, peer group mean bias: + 4.20 ± 1.44%) met NCEP recommendations according to which bias should be less than 3%. Only the methods using a non phenolic chromogen with a spectrophotometric detection met these recommendations (10% of participants, peer group mean bias: + 1.39 ± 2.75%).ConclusionsAs all three peer groups provided positively biased results, the consensus mean usually used to assess the trueness of routine methods is biased as well, which results in an erroneous estimation of method bias. Therefore, this study highlights the value added by reference method target values to assess trueness of field methods and monitor performance of clinical laboratories.     

Regulation of cell surface transferrin receptor-2 by iron-dependent cleavage and release of a soluble form

Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency.     

Long-term nephrotoxicity of cisplatin, ifosfamide, and methotrexate in osteosarcoma

The acute renal effects of chemotherapy are known, but long-term nephrotoxicity has rarely been investigated. The aim of the present study was to assess long-term renal function in children and adolescents who received at-risk chemotherapy, including cisplatin, ifosfamide, and methotrexate, to treat an osteosarcoma. Renal function tests [creatinine clearance, microalbuminuria, and renal excretion of sodium, potassium, chloride, calcium, magnesium (Mg), phosphorus (P), and uric acid] were prospectively performed 5.4±2.2 (±SD) years after chemotherapy (total cumulative dose: methotrexate 41±31 g/m², ifosfamide 39±14 g/m², cisplatin 674±188 mg/m²) in 18 children and adolescents. The results were compared with 13 normal volunteers matched for age and sex. Creatinine clearance, which was greater than 80 ml/min per 1.73 m² in all patients, correlated with the total dose of ifosfamide (r=0.55, P<0.05) and cisplatin (r=0.48, P<0.05). Microalbuminuria was noted in 4 patients. Hypomagnesemia was present in 4 and hypercalciuria in 3 patients; renal excretion of P, Mg, and uric acid was higher in patients than in controls. Glomerular function was not significantly altered and only mild tubular dysfunction was present. Since renal excretion of P and Mg were increased in patients compared with normal volunteers and hypercalciuria was occasionally seen, divalent ion disorders are the most-likely potential complications. Received September 29, 1997; received in revised form December 12, 1997; accepted February 18, 1998     

Phagocytic and Metabolic Activities of Macrophages from Mummichog Naturally Exposed to Pulp Mill Effluents in the Miramichi River

Chemicals present in bleached kraft pulp mill effluents (BKMES) can modulate the immune response of fish. This work was conducted to determine if mummichogs living downstream from a bleached kraft pulp mill (BKM) had an impaired immune response. Head kidney phagocytes from males and females were tested for their phagocytic and metabolic activities. Mummichogs were captured 4 km (S2) and 39 km (S1) downstream from a BKM in the Miramichi estuary (New Brunswick, Canada) and at two sites located upstream (S3) and 20 km downstream (S4) in a reference estuary, the Bouctouche. Both male and female mummichogs captured at S2 in the Miramichi estuary had a lower phagocytic activity, whereas only females from the same site had a decreased oxidative burst response, indicating a difference between sexes in the susceptibility to pollutants present in the Miramichi. Therefore, the results indicate that mummichogs exposed to BKMES have an impaired immune response and that females are more sensitive than males to the chemicals present in BKMES.     

Antihypertensive,vasodilating, and sympatholytic activities of ofornine® in spontaneously hypertensive rats

This study of spontaneously hypertensive (SH) rats documents oral antihypertensive activity of the vasodilator Ofornine®, an anthranilamide whose effect was sustained, at least in part, by suppression of compensatory renal and neurohumoral responses. Doses of 2.5 to 100 μg/kg p.0. reduced systolic blood pressure (BP) by up to 75 mm Hg. Effect lasted 6-24 hr and did not diminish during a 16 day (25 μg/kg/day) regimen. Metoclopramide (10.0 μg/kg)and haloperidol (3.0 μg/kg) antagonized the antihypertensive effect. Antihypertensive activity was confirmed by direct (arterial cannula) monitoring where 0.001-1.O μg/kg i.v. reduced mean BP by 6-47% without tachycardia. Doses of 1-10 ag/kg i.a. reduced perfusion pressure of the SH rat hindquarters by 35-74 mm Hg. A dose of 5.0 μg/kg p.0. reduced BP and urine norepinephrine levels of SH rats by 58 mm Hg and approximately 50% without raising plasma renin activity or causing sodium retention. How- ever, Ofornine® (25 μg/kg p.0. daily for 6 days) did not affect reflex bradycardic or tachy- cardic effects of phenylephrine and sodium nitroprusside in SH rats. These and other results indicate that Ofornine@ reduces SH rat blood pressure through vasodilating and presynaptic adrenolytic activities, and that a dopaminergic mechanism may be involved.     

Urethroplasty of female distal urethra

Results of experiences with urethroplasty for the repair of urethral meatal stricture of the female are reported. Discussion of the indications, results of the surgery, and a five-year follow-up study of the procedure is presented. Results are satisfactory, especially in children. A study of the resected pathologic specimens demonstrates inflammation rather than fibrosis is the basic cause of urethral meatal stricture.     

Ropivacaine decreases inflammation in experimental endotoxin-induced lung injury

BACKGROUND: Endotoxin causes acute lung injury, which can lead to acute respiratory distress syndrome. Because local anesthetics are known to attenuate inflammatory reactions, ropivacaine was tested for its possible antiinflammatory effect in lipopolysaccharide-induced lung injury in rat alveolar epithelial cells (AECs) and rat pulmonary artery endothelial cells (RPAECs) in vitro and in vivo. METHODS: AECs and RPAECs were stimulated for 4 h with lipopolysaccharide or lipopolysaccharide and 1 mum ropivacaine. Messenger RNA (mRNA) for intercellular adhesion molecule 1 was assessed. Isolated neutrophils were incubated with stimulated target cells to quantify adhesion and neutrophil-induced cytotoxicity in AECs and RPAECs. In vivo, lipopolysaccharide was instilled intratracheally with or without 1 mm intratracheally or intravenously administered ropivacaine. Bronchoalveolar lavage was performed 5 h later to determine neutrophil and albumin content, as well as concentrations of inflammatory mediators. RESULTS: In AECs and RPAECs, ropivacaine attenuated lipopolysaccharide-induced up-regulation of mRNA for intercellular adhesion molecule 1 by 41% and 24%, respectively (P < 0.05). In the presence of ropivacaine, increased neutrophil adhesion was down-regulated by 58% and 44% (P < 0.005), whereas cytotoxicity in AECs and RPAECs was diminished by 28% and 33%, respectively (P < 0.05). Enhanced neutrophil count in lipopolysaccharide lungs was reduced by 56% in the presence of intratracheally instilled ropivacaine (81% with intravenous ropivacaine; P < 0.005). Albumin was decreased by 46% with intratracheal ropivacaine (38% with intravenous ropivacaine; P < 0.05), and inflammatory mediators were decreased by 48-59% (69-81% with intravenous ropivacaine; P < 0.01). CONCLUSIONS: Ropivacaine intervention substantially attenuated the inflammatory response in acute lung injury and thus may carry an interesting potential for antiinflammatory treatment.     

Optimal use of peritoneal dialysis fluids in type 2 diabetes mellitus patients

The glucose side-effects, the main osmotic agent in conventional peritoneal dialysis (PD) solutions, are structural and functional changes of the peritoneal membrane, especially diabetic alterations in the microvasculature. Therefore, hyperpermeability with high small solutes transport and less ultrafiltration necessitates more and more high glucose concentration solutions. Glucose degradation products (PDF) and advanced glycation end-products (AGE) are formed and may induce peritoneal membrane alterations. More biocompatible solutions have to be used with less PDF and physiological pH. Icodextrin containing PD solutions have beneficial effect on sustained ultrafiltration for long dwells in PD, limitating fluid overload common in PD patients above all during peritonitis episodes. Amino acid-based PD solutions contribute to the prevention of malnutrition often observed in the diabetic PD population.