Validation of serum free light chain reference ranges in primary care.

BACKGROUND: The demand for measurement of serum immunoglobulin free kappa (κ) and lambda (λ) light chains has increased. The κ:λ ratio is used to assist in diagnosis/monitoring of plasma cell disorders. The binding site reference range for serum-free light chain κ:λ ratios of 0.26-1.65 was derived from healthy volunteers. Subsequently, a reference range of 0.37-3.1 for patients with chronic kidney disease has been proposed. Elevated free light chain concentrations and borderline raised free light chain ratios also may be found in polyclonal gammopathies and with other non-renal illnesses. This assessment was conducted to validate the established free light chain reference ranges in individuals from primary care. METHOD: A total of 130 samples were identified from routine blood samples collected in primary care for routine biochemistry testing and estimated glomerular filtration rate calculation. RESULTS: The median and range of κ:λ ratios found in each estimated glomerular filtration rate group used for chronic kidney disease classification were higher than previously described. This was the case for individuals with normal or essentially normal renal function with estimated glomerular filtration rates>90, (0.58-1.76) and estimated glomerular filtration rate of 60-90 mL/min/1.73 m(2), (0.71-1.93). Individuals with estimated glomerular filtration rate 15-30, (0.72-4.50) and estimated glomerular filtration rate <15 ml/min/1.73 m(2) (0.71-4.95) also had higher values when compared to the current renal reference range of 0.37-3.10. CONCLUSIONS: Elevation of free light chain-κ:λ ratios may occur in the absence of a reduced renal function shown by a normal estimated glomerular filtration rate and in the presence of reduced renal function by estimated glomerular filtration rate when comparing results with the established reference ranges. Explanations include choice of analytical systems or the presence of other concurrent non-plasma cell illness.

Zinc induced damage to kidney proximal tubular cells: studies on chemical speciation leading to a mechanism of damage.

UNLABELLED: This study was carried out to investigate whether zinc can potentiate renal toxicity using monolayer cultures of kidney proximal tubular cells and if so to establish the chemical species and the mechanism involved. METHODS: Zinc was prepared as the citrate complex at pH 7.4 in phosphate buffered saline. Monolayers of kidney proximal tubular cells under standard cell culture conditions were exposed to zinc concentrations of 0, 5 10, 20, 50 and 100 µmol/L. To assess cellular damage, thiazol blue (MTT) uptake, NAG and LDH release, DAPI staining and Tunel assay were used. Cytoprotective agents: trolox, cysteine, glutathione, ascorbic acid and sodium selenite were used to investigate if the damage was reversible. RESULTS: Incubation of kidney cells with zinc citrate showed a dose related reduction in cell viability (p<0.005) associated with cellular uptake of zinc ions. After 24 h incubation with 100 µmol/L Zn citrate, NAG release was not significantly different compared to the control whereas LDH increased 3 fold. DAPI staining showed apoptotic bodies within the cells confirmed by Tunel assay using flow cytometry. Electron microscopy showed significant morphological changes including loss of brush border, vacuolated cytoplasm and condensed nuclei. Trolox almost completely (>85±5%) and sodium selenite partially recovered (40±4%) the viability of cells exposed to Zn but no protection was observed with other cytoprotectants, e.g. glutathione, cysteine or ascorbic acid. In conclusion zinc can induce damage to kidney cells by a mechanism dependent on zinc ions entering the cell, binding to the cell organelles and disrupting cellular processes rather than damage initiated by free radical and ROS production.

Patient-reported fasting status for glucose-tolerance testing compared with fasting status identified by total bile-acid concentrations.

AIM: To investigate whether patients follow advice regarding the duration of a fast, prior to blood tests which require an overnight fast for their correct interpretation. METHOD: Patients referred for a glucose-tolerance test gave informed consent. They were asked to complete a questionnaire regarding the duration of their fast, and an additional blood sample was collected for fasting total bile-acid measurement. RESULTS: 184 patients were recruited. The median duration of fast self reported by patients was 14 h (range 12-26 h). Total bile-acid concentrations were within the fasting reference range except for six individuals. The median total bile-acid concentration found was 2.1 µmol/l (range 0.1-19.5 µmol/l). CONCLUSION: All patients who self-reported a duration of fast reported a fast recognised as being adequate for fasting blood tests. This was confirmed in almost all by low normal total bile-acid measurements, which were well within the fasting range.

A study on the stability of urinary free catecholamines and free methyl-derivatives at different pH, temperature and time of storage.

BACKGROUND: The goal of our study was to test the relative stability of urine, unconjugated, free catecholamines and the methyl derivatives. We measured the change in concentrations in commercially available urines after storage at various pH values, temperatures and time, from days up to 10 weeks. METHODS: Samples of commercial control urines were adjusted to pH 2.0, 4.0, 6.0 and 8.0 and aliquots stored at ambient temperature (20-26 degrees C), 4 degrees C and -18(o)C. The free catecholamines (cats) and the free methyl derivatives (mets) were measured after 1, 2, 3 and 6 days and 1, 2, 3 and 10 weeks using the automated sample trace enrichment dialysis (ASTED) procedure with reversed phase ion pair high performance liquid chromatography (HPLC) and coulometric detection. RESULTS: Free catecholamines were relatively stable, with <15% loss of concentration, when stored at pH 6.0 or less for at least 4 days and up to 10 weeks at pH 2.0 at either 4(o)C or -18(o)C. At pH 8.0, the concentration fell to <60% after 48 h and at a pH of 6.0 or 8.0, up to 90% was lost within the first week at 4(o)C and 25(o)C. More than 40% of free normetadrenaline and metadrenaline were lost after 1-2 weeks when stored at 20-25(o)C and pH 8.0. After 10 weeks at pH 4.0, 6.0 and 8.0, up to 90% loss was observed at 25(o)C. Free cats were stable at pH 2.0 and 4.0 at -18(o)C and the free mets were stable at -18(o)C over the entire time period studied and at all pHs. CONCLUSIONS: In the analysis of free catecholamine and the free methyl derivatives, urine samples should be acidified to a pH range 2.0-3.0 to ensure stability and hence the correct analysis.

Aluminium-induced injury to kidney proximal tubular cells: Effects on markers of oxidative damage.

Aluminium (Al)-induced injury to various cells including brain and bone is well described. We have previously shown that Al initiated damage to kidney cells in culture assessed by loss of cell viability, enzyme release and damage to cell brush borders. However, little is known about the mechanism(s) of these effects, we therefore investigated whether lipid peroxidation and/or sulphydryl depletion, i.e. cellular glutathione (GSH) depletion, could be part of this process(es). Monolayers of porcine kidney proximal tubular cells (PTC), LLC-PK1, were either exposed to 100 micromol/L Al as a citrate complex or cis-platin (cis-Pt) or mercury (Hg) as damage mediating controls (positive), or no added metals (negative control). Malondialdehyde (MDA), a marker of lipid peroxidation, cellular GSH as the intracellular sulphydryl compound, heat shock protein 70 (Hsp70), glutathione peroxidase (GPx) as a reactive oxygen species scavenger enzyme and cellular uptake of Al were assessed. MDA increased significantly (p = 0.03) more than two-fold in the cell lysate after PTCs were exposed to Al for 48 h. In Al-treated PTC, the GSH content of the cell lysate increased to 1.042 +/- 0.080 mmol/L compared with the 0.80 +/- 0.1.64 mmol/L control value. The Hsp70 content of the cells showed no significant change for Al (6.5% vs. 4.4% control value) and GPx activity increased slightly from 0.41 to 0.45 mU/mg protein. Treatment with cis-Pt and Hg also resulted in a significant increase in MDA and Hsp70 and reduction in cellular GSH and GPx activity. The low cellular Al uptake as well as some inherent insensitivity related to the cell type might be contributory factors for the small effect of Al on kidney cells and markers of oxidative damage.

Toxicological effects of iron on intestinal cells.

The aim of the present study was to investigate whether iron, which is involved in the formation of free radicals in vitro, can initiate cellular injury in human intestinal cells. The effects of various concentrations of iron were studied in preconfluent, colonic-cancerogenous cells, and also in postconfluent, differentiating cells. Cellular damage was assessed using cell proliferation (serial cell counting), tetrazolium dye (MTT) uptake, lactate dehydrogenase (LDH) release and apoptosis studies based on caspase-3 activities. Also the activities of the major antioxidative enzymes, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were measured after the cells had been exposed to iron. Our results indicated that preconfluent cells were more susceptible to iron toxicity, as assessed by a significant reduction in cell proliferation and MTT uptake in a concentration-dependent manner compared to the control. However, no evidence for MTT uptake was observed in postconfluent cells. Caspase-3 activity, an indicator of cell apoptosis, considerably increased in preconfluent cells at high iron levels compared to the control (p < 0.05), whereas postconfluent cells were not significantly affected. LDH release was similar for both groups and was significantly higher than the control at 900 microM iron and above. SOD activities were not affected by iron in either group, whereas GPx was considerably higher in iron-treated cells in both groups compared with the control (because of relatively high standard deviations this effect was not significant). In conclusion we suggest that iron exerts its toxic effects intracellularly especially in preconfluent Caco-2 cells, whereas only high iron doses were able to alter the viability of differentiating, enterocyte-like cells.

Toxic and biochemical effects of zinc in Caco-2 cells.

Zinc (in relatively high concentrations) can be toxic to intestinal cells. The aim of the present study was to quanitfy cellular injury in preconfluent, colonic cancerous cells and in postconfluent, differentiating human intestinal Caco-2 cells. Cellular damage was measured by using cell proliferation, lactate dehydrogenase (LDH)-release, and apoptosis studies. Furthermore, the activities of the major antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase] and differentiation markers (alkaline phosphatase and aminopeptidase-N) were determined after exposure of the cells to increasing amounts of zinc sulfate. Proliferation and viability decreased in a concentration-dependent manner. A noticeable increase of LDH-release correlated to cell rounding and detachment at relatively high zinc levels (200 muM) was observed in both groups of cells. Above 100 muM of zinc, significant apoptotic activity was found in the preconfluent cells. Zinc supplementation did not alter SOD activities. However, GPx and, in part, catalase activities tended to be higher in zinc-treated cells (nevertheless the results were not significant). Differentiation markers were noticeably induced by increasing amounts of zinc, especially in the preconfluent cells. In conclusion, we suggest that the susceptibility to zinc induced damage is equal in both confluentation groups of Caco-2 cells. Risk assessment for high concentrations seems recommendable.