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.

Electrophoretic separation of 5'-nucleotidase multiple forms.

A procedure is proposed for the separation of multiple forms of 5'-nucleotidase (EC 3.1.3.5) by cellulose acetate electrophoresis. The effects of various treatments (wheat-germ lectin, neuraminidase, n-butanol, papain, Triton X-100 and precipitation of LDL and VLDL) on the electrophoretic pattern of 5'-nucleotidase and alkaline phosphatase were studied. In healthy controls, the presence of three fractions with alpha 1, alpha 2 and beta mobilities was observed, the latter being the major one. In different hepatobiliary diseases a close relationship between the presence of high molecular weight alkaline phosphatase and the increase in the ratio Total/beta 5'-nucleotidase was observed, showing that the increase in total 5'-nucleotidase in these patients is mainly due to the alpha 1 isoform. The nature of these forms is discussed.

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.

Urinary D-glucaric acid and serum hepatic enzyme levels in chronic alcoholics.

Urinary D-glucaric acid (DGA) and the activities of gamma-glutamyl transferase (GGT) and other hepatic enzymes in serum were determined in 33 noncirrhotic male alcoholics who had continued to consume alcohol until at least 24 h prior to the taking of samples. DGA excretion was significantly greater in them than in a group of 30 healthy controls (p less than 0.001), exceeding the upper reference level in 38% of the alcoholic cases (as compared with 88% for GGT). In the alcoholic patients, there was highly significant correlation between urinary DGA and serum GGT (r = 0.613, p less than 0.001), suggesting that in both cases the increased levels are due to enzyme induction. None of the biochemical variables studied were significantly correlated with estimated daily alcohol consumption. Urinary DGA levels fell off rapidly with abstinence, and in 31 alcoholic patients who had consumed no alcohol for 5 days, there was no statistically significant correlation between DGA excretion and serum GGT (r = 0.158, p congruent to 0.4).

Cord serum gamma glutamyltransferase in newborns.

This article reports correlations among gamma-glutamyltransferase (GGT), fetal haemoglobin (fH), alpha-fetoprotein, 5'-nucleotidase, ceruloplasmin, and direct, indirect, and total bilirubin in the serum of blood taken from the umbilical cords of 128 newborns delivered after 37-42 weeks of gestation. GGT was significantly correlated with alpha-fetoprotein, but not with direct bilirubin, indirect bilirubin, total bilirubin, fH, or %fH. Neither fH nor %fH were correlated with alpha-fetoprotein, but there was highly significant negative correlation between both fH and %fH on the one hand, and gestational age and weight at birth on the other. The %fH was also correlated negatively with ceruloplasmin, which in turn exhibited negative correlation with alpha-fetoprotein. The predominant forms of GGT in umbilical cord and adult sera were, respectively, those with alpha 1 and alpha 2 mobility. In cord sera, delipidation with n-butanol brought about loss of GGT activity and a shift from an alpha 1 to an alpha 2 position, whereas no significant effect of this kind was observed in adult sera. Affinity chromatography through Concanavalin A-Sepharose showed cord sera to contain a proportion of bound-GGT (68.5 +/- 5.5%) that was significantly greater (p less than 0.001) than that found in adult sera (59.8 +/- 10.2%). It is concluded that the high GGT activity of cord sera is probably due to hepatic immaturity rather than maternal sources, enzymatic induction or microsomal lesions; that the predominant form of GGT in cord serum may be a complex with HDL and less sialized than the adult enzyme; and that, of the factors examined, the best indicator of neonatal maturity is fetal haemoglobin.

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.

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.

Carnitine deficiency associated with anticonvulsant therapy.

Valproic acid therapy is known to be associated with carnitine deficiency in adult as well as young epileptic patients. In a study of the possible existence of such side-effects with other anticonvulsants, 76.5% of adult patients treated with valproate were deficient in serum free carnitine, with acylcarnitine levels significantly higher than in controls (p less than 0.01), while the carnitine deficiency rate in a group of patients treated with anticonvulsants other than valproate was 21.5%. Since in clinical practice only about one fifth of patients are treated with valproate, this means that about 15% of epileptics are carnitine deficient because of valproate treatment and 17% because of other anticonvulsants. The mechanisms and clinical and biological consequences of the carnitine deficiency associated with antiepileptic drugs other than valproate are not known.

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.

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.

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.

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.

A de-sialylated isoform of serum 5'-nucleotidase: clinical and biological significance in hepatobiliary disease.

Using an electrophoretic technique on cellulose acetate, three multiple forms of the 5'-nucleotidase (5'NU) with mobilities alpha 1, alpha 2, and beta appear in the serum of healthy subjects. The difference between the alpha 1 and alpha 2 isoforms lies in their sialylation degree, the alpha 2-5'NU being a de-sialylated form. In 147 patients with different hepatobiliary diseases the alpha 2 isoform was present in only 19% of cases, and there was no significant difference in the activity of total 5'NU, alpha 1-5'NU and beta-5'NU between patients with or without alpha 2-5'NU, alpha 1-5'NU and beta-5'NU (P < 0.001), as with other biochemical indicators of liver damage. It is suggested that in hepatobiliary diseases an increase of the sialylation of serum 5'NU occurs, which would explain the absence of the desialylated alpha 2 isoform in the majority of cases. However, the decrease of hepatic receptors of asialoglycoproteins would lead to an increase of this de-sialylated isoform in the serum of certain patients.

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.