Diagnostic accuracy of a hyperbolic model in predicting digoxin concentrations based on glomerular filtration rates.

BACKGROUND: Inappropriate doses and high serum concentrations of digoxin are highly prevalent in patients with renal impairment, and the drug dosage adjustment according to the glomerular filtration rate (GFR) is recommended. The aim of our study was to evaluate the dependence degree of digoxin total clearance (CL) on GFR, and the diagnostic efficiency of a predictive model for serum digoxin steady-state concentrations (Css) from estimated GFR by Cockcroft-Gault formula. METHODS: In 400 outpatients treated orally with digoxin, serum Css were determined (fluorescence polarization immunoassay from Abbott Laboratories), and total CL was calculated. The prediction of Css was carried out using a hyperbolic model developed by Konishi et al in Japan (J Clin Pharm Ther 2002;27:257), and the constants of the equation were modified for a Caucasian population. RESULTS: Only 26% of the digoxin CL interindividual variability may be explained by differences in GFR, and this fact is a serious limitation for the derived predictive models. A 65% diagnostic efficiency was obtained for original and modified hyperbolic models in the correct classification of predicted Css as subtherapeutic, therapeutic or supratherapeutic with respect to obtained Css concentrations. CONCLUSIONS: The diagnostic efficiency obtained in the prediction of serum digoxin concentrations from estimated GFR values is unacceptable for the drug dosage adjustment in clinical practice.

Eficiencia diagnóstica de la predicción de concentraciones de digoxina en función de la tasa de filtración glomerular mediante un modelo hiperbólico / Diagnostic accuracy of a hyperbolic model in predicting digoxin concentrations based on glomerular filtration rates

Objetivos: Los pacientes con insuficiencia renal presentan una prevalencia elevada de dosis inapropiadas y de concentraciones elevadas de digoxina, y se ha recomendado el ajuste de la dosificación en función de la tasa de filtración glomerular (TFG). El objetivo de nuestro estudio fue evaluar el grado de dependencia con respecto a la TFG del aclaramiento total (CL) de digoxina, y la eficiencia diagnóstica de un modelo predictivo para la concentración sérica de digoxina en el estado de equilibrio (Css) en función de la TFG estimada por la ecuación de Cockcroft-Gault. Métodos: En 400 pacientes ambulatorios tratados por vía oral con digoxina se determinaron las Css séricas (inmunoanálisis de polarización de fluorescencia, Abbott Laboratories), calculandose el CL total. La predicción de Css se hizo mediante un modelo hiperbólico desarrollado por Konishi, et al. (J Clin Pharm Ther 2002;27:257), y las constantes de la ecuación fueron modificadas para la población caucasiana. Resultados: Sólo el 26% de la variabilidad interindividual del CL de digoxina puede ser explicado por diferencias de la TFG, y este hecho constituye una seria limitación para los modelos predictivos derivados. Se obtuvo una eficiencia diagnóstica del 65% para los modelos predictivos original y modificado en la clasificación correcta de las Css predichas como subterapéuticas, terapéuticas o supraterapéuticas con respecto a las Css obtenidas. Conclusiones: La eficiencia diagnóstica obtenida en la predicción de las concentraciones séricas de digoxina en función de los valores estimados de TFG es inaceptable para el ajuste de la dosificación del fármaco en la práctica clínica (AU)

Background: Inappropriate doses and high serum concentrations of digoxin are highly prevalent in patients with renal impairment, and the drug dosage adjustment according to the glomerular filtration rate (GFR) is recommended. The aim of our study was to evaluate the dependence degree of digoxin total clearance (CL) on GFR, and the diagnostic efficiency of a predictive model for serum digoxin steady-state concentrations (Css) from estimated GFR by Cockcroft-Gault formula. Methods: In 400 outpatients treated orally with digoxin, serum Css were determined (fluorescence polarization immunoassay from Abbott Laboratories), and total CL was calculated. The prediction of Css was carried out using a hyperbolic model developed by Konishi et al in Japan (J Clin Pharm Ther 2002;27:257), and the constants of the equation were modified for a Caucasian population. Results: Only 26% of the digoxin CL interindividual variability may be explained by differences in GFR, and this fact is a serious limitation for the derived predictive models. A 65% diagnostic efficiency was obtained for original and modified hyperbolic models in the correct classification of predicted Css as subtherapeutic, therapeutic or supratherapeutic with respect to obtained Css concentrations. Conclusions: The diagnostic efficiency obtained in the prediction of serum digoxin concentrations from estimated GFR values is unacceptable for the drug dosage adjustment in clinical practice (AU)

Two sampling time profiles for the abbreviated estimation of mycophenolic acid area under the curve in adult renal transplant recipients treated with mycophenolate mofetil and concomitant tacrolimus.

OBJECTIVE: Various studies have revealed that mycophenolic acid (MPA) area under the time concentration curve (AUC) may have clinical value in mycophenolate mofetil dose adjustment. As the full AUC measurement is impractical in clinical practice, several abbreviated AUC profiles using pre-dose, and two or three post-dose samples have been proposed; however, the possible use of lower sampling time profiles has an unquestionable practical interest, and the aim of our study was the evaluation of several two-points algorithms using only one post-dose sample. PATIENTS AND METHODS: In 60 MPA concentration-time profiles from 37 adult renal transplant patients treated with mycophenolate mofetil and concomitant tacrolimus, the MPA AUC values were estimated using the three sampling time algorithm (pre-dose, one-half and 2 h post-dose)of Pawinski et al. (Clinical Chemistry 48, 2002, 1497), trapezoidal extrapolated procedure according to Hale et al. (Clinical Pharmacology Therapeutics 64, 1998, 672), and two-points algorithm (pre-dose and 2 h post-dose) proposed by David-Neto et al. (Clinical Transplantation 19, 2005, 19). RESULTS: The AUC values estimated using the algorithm of Pawinski et al. had a very high correlation(r = 0.997, P < 0.001) with the trapezoidal extrapolated AUC results. The estimated AUC values obtained using the two-points algorithm of David-Neto et al. present a high correlation (r = 0.930, P < 0.001), acceptable mean prediction error (+3.3 +/- 1.8%), and a diagnostic efficiency of 94% in the classification of subtherapeutic, therapeutic, and supratherapeutic values, with respect to the three-points algorithm of Pawinskiet al. CONCLUSION: The two sampling time algorithm of David-Neto gave similar results to those of the three-sampling time algorithm of Pawinski, and both, with sampling over 2 h, may be useful for routine MPA AUC estimation in renal transplant recipients with concomitant tacrolimus. Both are unsuitable when unusually unpredictable pharmacokinetics are expected such as with entericcoated formulations.

Biochemical characterization of the GM2 gangliosidosis B1 variant.

The deficiency of the A isoenzyme of beta-hexosaminidase (Hex) produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease) has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age) with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphabeta heterodimer Hex A means that its activation energy (41.8 kJ/mol) is significantly lower than that of the betabeta homodimer Hex B (75.1 kJ/mol); however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

Biochemical characterization of the GM2 gangliosidosis B1 variant

The deficiency of the A isoenzyme of á-hexosaminidase (Hex) produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease) has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age) with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-á-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphaá heterodimer Hex A means that its activation energy (41.8 kJ/mol) is significantly lower than that of the áá homodimer Hex B (75.1 kJ/mol); however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

Assessment of copper status in pregnancy by means of determining the specific oxidase activity of ceruloplasmin.

BACKGROUND: Conditions not directly related to copper nutriture, such as pregnancy, infections and inflammation, which increase serum copper concentration even during copper deprivation, may be expected to conceal changes in copper status. It has been suggested that the specific enzymatic activity of ceruloplasmin (activity per unit mass of enzyme protein) may be a sensitive indicator of copper status and is not affected by factors such as hormones or sex. In this study, we investigated the behaviour of specific oxidase activity of ceruloplasmin and the copper/ceruloplasmin ratio in pregnant women. METHODS: Copper, immunoreactive ceruloplasmin and its oxidase activity were determined in serum from 52 women in the last trimester of normal pregnancy, and in 50 control women of similar age living in the same area and who were not taking oral contraceptives. The results are expressed as mean+/-S.E.M. RESULTS: In the group of pregnant women, significantly higher serum levels of copper, ceruloplasmin and its oxidase activity were found than in the control group (p < 0.001). In both groups, a high correlation was found between these biochemical variables (r > or =0.905, p < 0.001). However, in the group of pregnant women the specific oxidase activity for ceruloplasmin (364.4+/-3.3 vs. 407.5+/-3.8 U/g) and the copper/ceruloplasmin ratio (2.82+/-0.03 vs. 3.19+/-0.04 microg/mg) were significantly lower than in the control group (p < 0.001). CONCLUSIONS: Although pregnancy accelerates the rate of ceruloplasmin protein synthesis and release with an increase of serum copper, the decrease in specific oxidase activity of circulating ceruloplasmin would be an indicator of the degree of depletion of the mother's copper deposits in order to deal with the foetus' needs.

Serum cystatin C-immunoglobulin high-molecular-weight complexes in kidney and liver transplant patients.

BACKGROUND: It has been suggested recently that the glomerular filtration rate (GFR) in renal transplant patients is underestimated by serum cystatin C due to an impaired filtration of complexed cystatin C with immunoglobulins. Consequently, serum cystatin C may not be a reliable marker of GFR in these patients. Our study was designed to determine whether this supposition is correct. METHODS: In 87 serum samples from patients with various kidney diseases, 182 samples from renal transplant patients, and 72 samples from liver transplant patients, the concentrations of cystatin C and creatinine were determined, as well as the residual concentration of cystatin C after precipitation of macromolecules with polyethylene glycol (PEG; 6000 molecular weight). RESULTS: The residual concentration of serum cystatin C after precipitation with PEG in all cases was much higher (70 to 100%) than that expected in the case of the existence of cystatin C-immunoglobulin complexes. In the kidney and liver transplant patients, there was no significant correlation between the residual concentration of cystatin C and the postoperative time (r = -0.098). CONCLUSIONS: The results suggest that in renal or liver transplant patients there is no formation of high molecular weight serum cystatin C-immunoglobulin complexes, regardless of the post-transplant period.

Immunochemical and enzymatic study of ceruloplasmin in rheumatoid arthritis.

Forty adult patients (30 women and 10 men) with rheumatoid arthritis (RA), treated with nonsteroidal anti-inflammatory drugs, were studied. Serum levels of immunoreactive ceruloplasmin, oxidase activity of the ceruloplasmin and total copper, as well as the specific oxidase activity (enzyme activity per unit of mass) and the copper/immunoreactive ceruloplasmin relationship were significantly higher in the group of patients than in the healthy control group (p < 0.001). However, no significant difference was found for the concentration of non-ceruloplasmin copper between both groups. A statistically significant negative correlation was obtained for the concentration of serum thiobarbituric acid-reacting substances with the immunoreactive ceruloplasmin and its oxidase activity in the group of patients (p < 0.005). These results suggest that in RA increases of serum copper are produced at the expense of the fraction linked to the ceruloplasmin, diminishing the proportion of apoceruloplasmin and other forms poor in copper. Although the increase in the serum concentration of ceruloplasmin might offer an additional safeguard against oxidative stress. it does not appear to have a beneficial effect upon the activity of the illness as evaluated by means the biological inflammation markers C-reactive protein, erythrocyte sedimentation rate and sialic acid.

Pharmacological modification of the serotonergic transmitter system and beta-N-acetylhexosaminidase activity in rats.

The enzyme activity and activation energy of plasma beta-N-acetylhexosaminidase (Hex) was determined in rats whose serotonergic system had been pharmacologically altered. In the group of animals treated with 5-hydroxytryptophan, in the different dissected brain regions (brain stem, cortex and hippocampus) significantly higher levels of serotonin and 5-hydroxyindolacetic acid were found, and significantly lower in the group treated with p-chlorophenylalanine, than in the control group. In the total number of animals studied (n = 21), a statistically significant correlation was found between the plasma concentration of 5-hydroxyindolacetic acid and the levels of this metabolite in the different brain regions (p < 0.001). No significant differences were found for the activity of Hex in the plasma, or for its activation energy, which is a marker of its isoenzyme composition, among the three groups of animals. The results obtained using our experimental model in rats do not confirm the hypothesis of other authors who suggest that the Hex responds secondary to increases or decreases of serotonin turnover, and could be a biological test to monitor the serotonin status in psychiatric patients.

Comparison of predose vs 2-h postdose blood metabolites/cyclosporine ratios in kidney and liver transplant patients.

OBJECTIVES: It has recently been suggested that when adjusting doses of cyclosporine (CsA), determining its concentration in blood samples taken 2 h postdose (C(2)) is more clinically beneficial than using the predose concentration (C(0)). We determined C(0) and C(2) concentrations of CsA and their metabolites in samples taken from nine kidney and seven liver transplant patients. Similarly, the so-called metabolic ratios (MR)-metabolites to CsA parent ratios-were calculated to characterise the most suitable moment of blood sampling for obtaining a greater analytical specificity with monoclonal immunoassays. METHODS: The determination of CsA and CsA + metabolites was made using the enzyme multiplied immunotechnique and the polyclonal fluorescence polarization immunoassay Abbott TDx, respectively. RESULTS: The poor correlation between C(0) and C(2) of CsA (n = 82, r = 0.387, p < 0.001) is greatly inferior to that obtained between C(0) and C(2) of metabolites (n = 82, r = 0.912, p < 0.001). A highly significant difference (p < 0.001) was found between MR(0) values (mean 2.87 +/- 0.12, median 2.48) and MR(2) values (mean 1.73 +/- 0.09, median 1.46), although there is a good correlation between them (r = 0.878, p < 0.001). CONCLUSIONS: The extent of the positive bias (deviation) of CsA immunoassays compared with the high-performance liquid chromatography results is related to the MR values. As the MR(2) values are significantly lower than the corresponding MR(0), in practice a greater analytical specificity would be obtained with the different monoclonal immunoassays in the determination of the 2 h postdose CsA concentration than in that of trough concentration.

Estudio comparativo de los resultados de digoxina sérica obtenidos mediante dos inmunoanálisis: efecto del material de calibración / Comparative study of serum digoxin results obtained by means two immunoassays: effects of the calibration materials

Se realizó un estudio comparativo entre dos métodos analíticos -inmunoanálisis de fluorescencia polarizada Abbott (FPIA) e inmunoturbidimetría Roche (INTB)- para la determinación de digoxina sérica. La precisión obtenida con ambas técnicas es análoga y la correlación entre los resultados es altamente significativa (r= 0.951, p<0.001). Sin embargo, la sobreestimación media de los resultados de la técnica INTB con respecto al FPIA es aproximadamente del 20 por ciento. Esta diferencia es debida, al menos parcialmente, a los materiales de calibración utilizados, aunque no existiría una heterogeneidad del analito o un efecto matriz (AU)

Plasma beta-N-acetylhexosaminidase isoenzyme composition and temperature conversion factors.

Using the chromogenic substrate 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide for the activity determination of plasma beta-N-acetylhexosaminidase (Hex), the temperature conversion factors (TCF) offer a highly significant positive correlation with the relative proportion of Hex B isoenzyme (P< 0.001). The calculation of TCF 37 degrees/25 degrees C allows the isoenzyme composition of Hex to be determined quickly and cheaply. The results may be superimposed over those obtained in a previously described method based on the calculation of the enzyme's activation energy using four temperatures. However, the use of TCF 37 degrees/30 degrees C does not appear to comply with the required demands.

Relationship between plasma ammonia concentration and beta-N-acetylhexosaminidase isoenzyme activities in liver cirrhosis.

Ammonia is known to increase the secretion of beta-N-acetylhexosaminidase (Hex) to the extracellular medium in cultured human fibroblasts and Hep G2 cells. We examined 35 patients with liver cirrhosis and the results revealed a significant increase for the plasma activities of total Hex and its isoenzymes Hex A and Hex B (p < 0.001). The partial correlations, with other biochemical markers of liver injury constant, between plasma ammonia concentration and the activity (r = 0.658) and the proportion in percentage of the Hex B isoenzyme (r = 0.692) were statistically significant (p < 0.001). The increased concentration of ammonia could explain, at least partially, an increased secretion of Hex B isoforms to the plasma in patients with liver disease.

Specific oxidase activity of cord serum ceruloplasmin in the newborn.

In a study of 50 full-term newborns, we found serum levels of ceruloplasmin, its oxidase activity and specific oxidase activity (activity per unit mass of enzyme protein) which were significantly lower than in adult subjects (p<0.001). A significant correlation was obtained between the specific oxidase activity of the neonatal ceruloplasmin and the transferrin/alpha-fetoprotein ratio (p<0.01), which reflects the maturity of the fetal hepatocyte through its ability to synthesise fetal- and non-fetal-associated proteins. The lower specific oxidase activity of the ceruloplasmin in newborns would be due to a higher relative proportion of apoprotein, and/or other molecular forms with a poor copper content in the oxidase sites. This could be due to an inability to transfer copper into a common intracellular pool for holoceruloplasmin synthesis and biliary copper excretion. A high interindividual variability was found for the specific oxidase activity of the neonatal ceruloplasmin, and in the newborns with higher levels of ceruloplasmin this, from a catalytic point of view, is more similar to the adult form.

Characterization of the isoenzyme profile of beta-N-acetylhexosaminidase in the urine of newborns.

The urinary isoenzymes of beta-N-acetylhexosaminidase (Hex) in newborn infants were characterised by chromatography, electrophoresis, thermodynamic analysis and through substrate specificity. No qualitative difference was found for the major Hex A and Hex B isoenzymes between full-term or premature newborns and adults, although in the latter group the relative proportion of Hex B is much lower (18.5 +/- 2.7% vs. 36.3 +/- 1.0%). An additional minor enzyme form was found in some premature newborns, which eluted from the DEAE-cellulose column at a higher concentration of NaCl than Hex A and, like this isoenzyme, is able to hydrolyse 4-methylumbellipheryl-2-acetamido-2-deoxy-beta-D-glucopyranoside-6 -sulphate, which would suggest that it has alpha subunits in its molecule. These results do not confirm the hypothesis of other authors about the existence of a unique fetal Hex isoenzyme in neonatal urine which eluted before the application of the NaCl gradient, similarly to the Hex B.

Thermodynamic characterisation of the mutated isoenzyme A of beta-N-acetylhexosaminidase in GM2-gangliosidosis B1 variant.

Here we report the determination of the activation energies of the plasma isoenzymes of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52), isolated by chromatography in DEAE-cellulose, using the neutral chromogenic substrate 3,3'dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide. The activation energy of mutated Hex A isoenzyme (Ea approximately 71.5 kJ/mol) from a patient with GM2-gangliosidosis B1 variant, homozygote for the G533-->A (Arg178His) mutation, was significantly higher than that of normal Hex A (Ea approximately 41.8 kJ/mol) and analogous to that of Hex B isoenzyme (Ea approximately 75.1 kJ/mol). The determination of this thermodynamic variable of Hex in different biological specimens could allow for a straightforward biochemical characterisation of the GM2-gangliosidosis B1 variant.

Assay of beta-N-acetylhexosaminidase isoenzymes in urine by means of determination of their activation energy without removing endogenous low-molecular-mass components.

The determination of the activation energy of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52), using 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide as substrate, allows its isoenzyme composition to be evaluated in different biological specimens. However, in the analysis of urine samples, it is necessary first to remove the endogenous low-molecular-mass components, as these provoke an over-estimation of the activation energy of the Hex and, consequently, of the relative proportion of Hex B isoenzyme. The study of this interference has allowed urea to be characterised as the only urinary metabolite that is responsible, and to establish a mathematical expression for the correction, in relation to the endogenous urea concentration, of the activation energy of the Hex obtained experimentally in samples of native urine. The results thus obtained for the isoenzyme composition of urinary Hex are similar to those found using an electrophoretic separation procedure.