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Biomedical subjects

K R Copeland

Publications and source records attributed to K R Copeland.

At least 19 recordsLinked to original sources
PubMed1998

New modified fluorescence polarization immunoassay does not falsely elevate vancomycin concentrations in patients with end-stage renal disease.

Recent literature has urged caution in the interpretation of vancomycin serum concentrations in patients with end-stage renal disease (ESRD), because falsely elevated levels in excess of 70% have been reported with the most commonly used fluorescence polarization immunoassay (FPIA). The purpose of this study was to evaluate the performance of a recently modified FPIA assay for use in patients with ESRD, in comparison to high-performance liquid chromatography (HPLC) and an enzyme-mediated immunoassay technique (EMIT). Serum vancomycin samples were prospectively collected from adults with ESRD undergoing chronic hemodialysis. Each sample was stored at -70 degrees C until analyzed in duplicate by FPIA, EMIT, and HPLC. In an in vitro experiment, blank serum samples with 15 microg/ml vancomycin were spiked with increasing amounts of CDP and analyzed in duplicate with the modified FPIA assay. When compared to HPLC, no statistically significant difference was found in patients with ESRD with the use of the modified FPIA assay (mean concentrations, HPLC 14.92 microg/ml, FPIA 15.96 microg/ml), with FPIA exhibiting a positive bias of 0.64 microg/ml and a precision of +/-3.49 microg/ml (n = 18, p = 0.44). The mean EMIT concentration was 18.34 microg/ml, with a positive bias of 3.43 microg/ml and a precision of +/-5.17 microg/ml (p < 0.01). The addition of increasing amounts of CDP to vancomycin in vitro resulted in concentrations similar to those expected in the absence of significant cross-reactivity with the modified FPIA assay. The modified FPIA assay is a satisfactory tool for monitoring vancomycin serum concentrations in patients with ESRD undergoing hemodialysis. Results obtained with EMIT were not as precise as with FPIA.

Adolescent↗
PubMed1996

Adaptation of a quantitative immunoassay for urine myoglobin. Predictor in detecting renal dysfunction.

Myoglobinuria, subsequent to rhabdomyolysis, may cause acute renal failure. For this reason, many qualitative and quantitative tests have been developed for the detection of myoglobin in urine. The authors describe the adaptation and optimization of the Stratus II serum myoglobin immunoassay to quantify urine myoglobin. In addition, the assay was used to accurately determine urine myoglobin concentrations in subjects at potential risk for myoglobin-induced renal dysfunction and the results obtained compared to conventional qualitative methods for urine myoglobin. The assay demonstrated with-in run and between-run coefficient of variations (CVs) of 6.2% and 7.2%, respectively, was linear from 0-950 micrograms/L, demonstrated good recovery, and was free from interference by hemoglobin, creatinine, and urea. Specimens were diluted with 0.1 mol/L phosphate buffer, pH 9.0 containing 3% bovine serum albumin before analysis. Myoglobin was assayed on urine obtained from 30 patients suspected of having myoglobinuria. Fifteen of 17 patients with serum creatinine greater than 1.4 mg/dL had myoglobin concentrations greater than 20,000 micrograms/L, whereas the remaining 31 patients with normal serum creatinine had urine myoglobin concentrations of less than 18,000 micrograms/L. If serum creatinine is used as an indicator of renal function, it would appear that accurate measurement of urine myoglobin may facilitate identification of patients with increased susceptibility to myoglobin-induced acute renal failure.

Acute Kidney Injury↗
PubMed1996

Ultrafiltration discrepancies in recovery of myoglobin from urine.

We investigated the efficiency, accuracy, and reliability of the ultrafiltration/dipstick methodology commonly used to diagnose myoglobinuria. Twenty-five myoglobin-containing urine specimens were filtered by centrifugation for 15 min at 1500g through a Centricon-30 membrane filter. Both the original specimen and filtrate were assayed for myoglobin. The amount of myoglobin recovered subsequent to filtration varied from <1-38%. This poor and variable recovery was independent of sample matrix or precentrifugation of the specimens. This was most critical for urine specimens with myoglobin concentrations <60 000 microg/L. Fourteen of 18 such filtrates had concentrations <350 microg/L, a concentration below which a negative result would be obtained by using conventional dipstick methods. Thus, the use of this procedure has the potential to misdiagnose patients with myoglobin concentrations associated with increased risk of subsequent renal dysfunction, in particular when urine myoglobin concentrations are <60 000 microg/L.

False Negative Reactions↗
PubMed1995

Single-strand conformational polymorphisms (SSCP): studies of the genetic polymorphisms of exon 4 of apolipoprotein C III.

OBJECTIVE: We used single-strand conformational polymorphism (SSCP). To screen for mutations/polymorphisms in exon 4 of the apolipoprotein C III in 45 patients with hypertriglyceridemia and 46 control individuals, single-strand conformational polymorphism was investigated using restriction endonuclease and amplification refractory mutations systems (ARMS). RESULTS: SSCP identified six patterns corresponding to six genotypes. We confirmed that the different genotypes result from the two polymorphic sites at positions 3175 and 3206 of the apo C III gene. Only three of four possible haplotypes were found in the study population. This resulted in the identification of 6 of the 10 possible genotypes. CONCLUSIONS: SSCP is a useful method to screen for both known and unknown mutations/polymorphisms and should have increasing applications in clinical laboratories involved with the study of genetic markers of a wide variety of diseases.

Adult↗
PubMed1995

Intracellular magnesium content of mononuclear blood cells and granulocytes isolated from leukemic, infected, and granulocyte colony-stimulating factor-treated patients.

The intracellular magnesium (Mg) concentration of granulocytes and mononuclear blood cells (MBCs) was determined in cells isolated from patients with several disorders. The mean (+/-SD) Mg content of MBCs isolated from patients diagnosed with lymphocytic leukemia, myelocytic leukemia, or infection; from patients treated with granulocyte colony-stimulating factor (G-CSF); and from healthy volunteers (control group) was 2.3 (+/-0.6), 3.3 (+/-0.5), 4.1 (+/-0.8), 3.9 (+/-0.4), and 3.9 (+/-0.6) fmol/cell, respectively. The Mg content of MBCs isolated from patients with lymphocytic and myelocytic leukemia, but not those from patients with infection or receiving G-CSF treatment, were significantly lower (P < 0.001) than those from the control subjects. The mean Mg concentration of granulocytes obtained from lymphocytic leukemia, myelocytic leukemia, infection, and G-CSF patients and from the control group was 3.2 (+/-0.9), 3.4 (+/-0.5), 3.8 (+/-0.6), 4.5 (+/-0.6), and 4.6 (+/-0.6) fmol/cell, respectively. Granulocytes isolated from leukemic and infectious patients yielded lower intracellular Mg concentrations (P < 0.005) than those from patients receiving G-CSF and the control group. This study demonstrates that intracellular Mg content is altered in several pathological states. Several factors, including depleted Mg stores or altered intracellular Mg binding sites, could be responsible for these changes. Apparently, intracellular Mg content may be of use in assessing total body Mg status.

Granulocyte Colony-Stimulating Factor↗
PubMed1994

Determination of a T/G polymorphism at nucleotide 3206 of the apolipoprotein C III gene by amplification refractory mutation system.

We used the amplification refractory mutation system (ARMS)--a polymerase-chain-reaction-based method--to determine the 3206 T-to-G polymorphism on exon 4 of the apolipoprotein (apo) C III gene. Apo C III is an inhibitor of the enzyme lipoprotein lipase (EC 3.1.1.34). Previous studies have demonstrated that a polymorphism at nucleotide 3175 on exon 4 of this gene is associated with hypertriglyceridemia. We studied 45 hypertriglyceridemic and 46 age-matched controls for the 3206 T-to-G polymorphism. The results showed a significant difference in the distribution of the genotypes with respect to this allele between the hypertriglyceridemic and control individuals. We also determined the presence of the SacI site at nucleotide 3175 in these same individuals and found no significant difference in SacI genotypes between the two groups. This study reaffirms the usefulness of ARMS as a simple, reliable method for detecting mutations and polymorphisms in clinical and epidemiological studies.

Adult↗
PubMed1993

Cyclosporin G and metabolite binding to cyclophilin and a 50-kDa binding protein related to in vitro immunosuppression.

Seven purified metabolites of cyclosporin G (CsG) were studied for binding to cyclophilin and a 50-kDa binding protein (50-kDa BP). The ratios of the metabolite dissociation constants with respect to CsG were compared with in vitro immunosuppression by using the primary mixed lymphocyte suppression assay. The immunosuppressive potency ratio of the parent compounds, both cyclosporin A (CsA) and CsG, compared favorably with the drug dissociation constants for cyclophilin and the 50-kDa BP. Three of the seven metabolites had comparable binding and potency ratios for the 50-kDa BP. In contrast, none of the seven metabolites appreciably bound to cyclophilin in the concentration range tested.

Amino Acid Isomerases↗
PubMed1993

Accuracy verification and identification of matrix effects. The College of American Pathologists' Protocol.

Proficiency testing using stabilized control materials has been used for decades as a means of monitoring and improving performance in the clinical laboratory. Often, the commonly used proficiency testing materials exhibit "matrix effects" that cause them to behave differently from fresh human specimens in certain clinical analytic systems. Because proficiency testing is the primary method in which regulatory agencies have chosen to evaluate clinical laboratory performance, the College of American Pathologists (CAP) has proposed guidelines for investigating the influence of matrix effects on their Survey results. The purpose of this investigation was to determine the feasibility, usefulness, and potential problems associated with this CAP Matrix Effect Analytical Protocol, in which fresh patient specimens and CAP proficiency specimens are analyzed simultaneously by a field method and a definitive, reference, or other comparative method. The optimal outcome would be that both the fresh human and CAP Survey specimens agree closely with the comparative method result. However, this was not always the case. Using several different analytic configurations, we were able to demonstrate matrix and calibration biases for several of the analytes investigated.

Bias↗
PubMed1992

Comparison of the effects of cyclosporine and its metabolites on the release of prostacyclin and endothelin from mesangial cells.

CsA-induced alterations in mesangial cell production of potent vasoactive substances may be a contributing factor to the decreased renal blood flow and glomerular thrombosis associated with CsA nephrotoxicity. In this study the toxic effects of several CsA metabolites, previously isolated and characterized by mass spectrometry and nuclear magnetic resonance, were investigated in cultured rabbit mesangial cells. AM4N and AM9 were the most cytotoxic metabolites examined, having potency ratios of 0.28-0.56 with respect to CsA for the inhibition of cell growth and DNA synthesis. In addition the effects of several CsA metabolites were examined on the basal release of prostacyclin and endothelin. A concentration-dependent decrease in prostacyclin production, as measured by release of its stable metabolite, 6-keto-PGF1 alpha, was observed in cells treated with CsA and metabolites. CsA metabolites were equipotent as parent drug resulting in a significant (P less than 0.05) 20-40% reduction in prostacyclin production. The dihydroxylated metabolites of CsA, AM19, and AM1c9 resulted in a significantly (P less than 0.05) increased production of endothelin from the cells. In contrast parent drug resulted in a slight decrease in its production. In general CsA metabolites were found to be significantly less cytotoxic than parent drug in mesangial cells. However, the finding that certain CsA metabolites could alter production of vasoactive substances from these cells suggests that CsA metabolites may directly alter the renal hemodynamics, producing alterations consistent to that observed for CsA-induced renal side effects in vivo.

Animals↗
PubMed1991

Correlation of cyclosporine and metabolite binding to cyclophilin and a 50 kDa binding protein with in vitro immunosuppression: a preliminary report.

Seven purified cyclosporine (CsA) metabolites were analyzed for binding to cyclophilin and to a 50 kDa protein purified from a JURKAT cell line. In addition, the potency of the seven metabolites, relative to CsA, was obtained using a primary mixed lymphocyte culture (MLC) suppression assay. CsA, M1, 17, and 21 were found to be immunosuppressive in the concentration range used (0-500 ng/mL). These results were then compared to protein binding. CsA and metabolite 17 (M17) bound to both proteins. Conversely, M1, 13, 21, and 26 bound only to cyclophilin, while M8 and M18 bound only to the 50 kDa protein.

Amino Acid Isomerases↗
PubMed1991

Cyclosporine nephrotoxicity--experimental models.

Cyclosporine A (CsA) represents one of the more important therapeutic advances in the field of kidney transplantation. However, its effectiveness is limited by serious side effects, most notably nephrotoxicity. Investigation of the mechanisms of CsA-induced renal dysfunction has been hampered by the lack of a suitable experimental model. The majority of studies using the rodent have failed to exhibit all of the structural changes seen in chronic CsA-induced nephrotoxicity reported in man, using pharmacologic doses administered orally, subcutaneously, or intravenously. More recently, studies using the rabbit as an experimental model have demonstrated leucocyte infiltration, tubular atrophy, interstitial fibrosis, and arteriolopathy after therapeutic doses of CsA over 30 days. These changes are similar to those seen in chronic CsA-induced nephrotoxicity in man.

Animals↗
PubMed1991

The isolation, structural characterization, and immunosuppressive activity of cyclosporin G (NVa2-cyclosporine) metabolites.

Seven cyclosporin G metabolites were isolated by high-performance liquid chromatography from the urine of normal subjects receiving the drug. The structure and purity of the metabolites were assessed by fast atom bombardment/mass spectroscopy, by proton nuclear magnetic resonance (NMR), and by 13C-NMR. The structural modifications of the cyclosporin G metabolites consisted primarily of hydroxylation and demethylation, as is the case for cyclosporin A metabolites. The immunosuppressive activities of the metabolites were tested in three separate in vitro systems: a primary and secondary mixed lymphocyte system, as well as a mitogen stimulated system. In general, the metabolites have immunosuppressive activity of less than 10% of cyclosporin G. The significance of these findings in relation to the therapeutic monitoring of cyclosporin G is discussed.

Chromatography, High Pressure Liquid↗
PubMed1991

Chronic ciclosporin nephrotoxicity: a rabbit model.

Ciclosporin (CsA) was administered intravenously for 30 days to New Zealand white rabbits at the following doses: group A, 2.5 mg/kg/day; group B, 5.0 mg/kg/day; group C, 10 mg/kg/day; group D, saline control; group E, vehicle (cremophor-EL) control. Creatinine clearance was significantly reduced (p less than 0.05) at time of sacrifice in group C as compared to controls. There were no apparent differences among the five groups with respect to body weight gain or blood pressure. Morphologically, there were marked changes in the cytoarchitecture of the kidneys from all groups of animals treated with CsA. At the light microscopic level, in contrast to controls, there was the presence of leukocyte infiltration, tubular atrophy, interstitial fibrosis, and arteriolopathy. At the ultrastructural level, numerous vacuoles, lysosomal-like structures, and loss of cellular integrity were seen in the proximal and distal tubules, as well as interstitial fibrosis in the form of numerous collagen fibers. No changes in the glomeruli were seen in any of the CsA-treated groups. These findings are consistent with chronic CsA nephrotoxicity similar to that seen in man.

Animals↗
PubMed1990

Toxicity of cyclosporine metabolites.

Eight cyclosporine (CsA) metabolites were isolated from the urine of renal transplant recipients. The structure and purity of the metabolites were characterized by fast atom bombardment/mass spectroscopy as well as by proton and 13C nuclear magnetic resonance. The in vitro toxicity of the metabolites were tested using a porcine renal epithelial cell line (LLC-PK1). None of the metabolites was as effective as CsA in inhibiting cell growth and DNA, RNA, or protein synthesis, with the majority of them exhibiting activity less than 10% of that of CsA when the IC50 (the concentration required for 50% inhibition of that particular metabolic function) values were compared. The exception to this was the demethylated metabolite M-21, which exhibited a potency of 17-50% of CsA for the various metabolic parameters examined. The results suggest that the immunosuppressive activity of metabolites may be dissociated from their toxicity. Morphologically, CsA and the metabolite M-21 resulted in changes consistent with the vacuolization seen in tubular cells exposed to CsA in vivo. In contrast, M-17 up to the maximum concentration tested (25,000 micrograms/L) was found not to cause such changes.

Cell Line↗
PubMed1990

Chronic hyperglycemia in experimental diabetes mellitus of short duration does not contribute to muscle capillary basement membrane thickening.

The effect of chronic hyperglycemia on the relationship of nonenzymatic glycation and capillary basement membrane thickness in muscle was studied in streptozotocin-induced diabetic rats early in the course of diabetes mellitus. Diabetic animals were placed on either standard (24%) or restricted (8%) protein diet. The animals on 8% protein diet had elevated glycated hemoglobin levels (p less than 0.01) and increased levels of nonenzymatic glycation of basement membrane (p less than 0.01) as compared to insulin-treated diabetic (euglycemic), age-matched control, and streptozotocin-injected nondiabetic animals also on 8% protein diet. In contrast, diabetic animals on restricted (8%) protein diet and those on standard (24%) protein diet showed no statistical differences between them with regards to the above parameters. Moreover, there were no statistical differences among diabetic and control animals on either 8 or 24% protein diet with respect to muscle capillary membrane thickness. Even though the peripheral muscle biopsy study of capillary basement membrane is less invasive than kidney biopsy, the results of this study suggest that neither nonenzymatic glycation nor basement membrane thickness can be utilized as predictors of renal dysfunction during early onset of diabetes mellitus.

Animals↗
PubMed1990

Immunosuppressive activity of cyclosporine metabolites compared and characterized by mass spectroscopy and nuclear magnetic resonance.

Eight cyclosporine (CsA) metabolites were isolated from the urine of renal-transplant patients by high-pressure liquid chromatography. Structure and purity of the metabolites were assessed by fast atomic bombardment/mass spectroscopy, by proton nuclear magnetic resonance (NMR), and, when the quantity of metabolites permitted, by 13C-NMR. The immunosuppressive activities (I) of the metabolites were tested in three separate in vitro systems: primary and secondary mixed lymphocyte reactions as well as by a mitogen-stimulated system. The I, as measured by comparing the concentration of each metabolite required for 50% inhibition of incorporation of [3H] thymidine, varied among the assay systems, as did the ranking of I among the test systems. In general, the I of most metabolites in all assay systems were less than 10% of that for CsA. Metabolites with single modifications exhibited the greatest I; e.g., that of M-17 was congruent to 16% of that of CsA (potency ratio 0.16) in a secondary mixed lymphocyte reaction. The significance of these findings in relation to therapeutic monitoring of CsA is discussed.

Chromatography, High Pressure Liquid↗
PubMed1990

Abbott TDx monoclonal antibody assay evaluated for measuring cyclosporine in whole blood.

We report here the evaluation of the Abbott TDx assay with a monoclonal antibody for selectively quantifying cyclosporine (CsA) in whole blood. Over the clinically relevant concentration ranges, results with this assay demonstrated within- and between-run CVs of less than 2.5% and 5%, respectively; sensitivity of 25 micrograms/L; good analytical recovery (100.3%); and linearity with whole-blood specimens. The percentage cross-reactivity of the major CsA metabolites varied from 15.3% for AM9 (M-1), 8.2% for AM1 (M-17), and 3.7% for AM4N (M-21), to less than 3% for the other metabolites tested. Results with the TDx assay (y) correlated well with those by the Sandimmune selective RIA (x; Sandoz) with blood specimens from 44 renal-transplant recipients (n = 44, x= 187.3, y = 198.9, y = 5.49 + 1.03x, r = 0.987). The TDx values were on average 24% higher than those by HPLC (x') with the same patients' specimens (n = 44, x' = 159.9, y = 198.9, y = 15.9 + 1.14x', r = 0.967). We conclude that the Abbott TDx monoclonal antibody assay provides a rapid, precise, and accurate means for quantifying CsA in whole blood.

Antibodies, Monoclonal↗