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Background: It has recently been reported that patient selection has a strong impact on the agreement between glomerular filtration rate (GFR) estimates from serum cystatin C and creatinine. The aim of our study was to evaluate the effect of creatinine production rate (CPR) on this subject., Material and Methods: GFR was estimated from serum cystatin C and from creatinine using the 4- and 6-variable Modification of Diet in Renal Disease (MDRD), and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations in 50 healthy subjects, 43 patients with renal failure, 794 kidney and 104 liver transplant recipients, 61 patients with heart failure, 59 patients with biliary obstruction, and 113 critically ill patients., Results: In the 295 patients with impaired CPR (< 900 mg/24 h/1.73 m(2)), discordances of more than 40% between GFR(MDRD4) and GFR(cystatinC) were observed in 38% of cases, between GFR(MDRD6) and GFR(cystatinC) in 22%, and between GFR(CKD-EPI) and GFR(cystatinC) in 27% (in all cases due to GFR overestimation from creatinine). In the 929 patients with maintained CPR (> 900 mg/24 h/1.73 m(2)), greater discordances than 40% between GFR(MDRD4) and GFR(cystatinC) were observed in 8% of cases, between GFR(MDRD6) and GFR(cystatinC) in 9%, and between GFR(CKD-EPI) and GFR(cystatinC) in 7% (in the major part of cases due to GFR overestimation from cystatin C)., Conclusion: The main source of differences of more than 40% between GFR estimates from serum creatinine and cystatin C is a GFR overestimation in patients with low CPR and GFR underestimation in patients with high CPR by the creatinine-derived equations.

Background: Pro-inflammatory cytokine production is directly inhibited by acetylcholine (ACh), and a relationship between total circulating ACh hydrolytic capacity and inflammatory reactions has been previously reported. Butyrylcholinesterase (BChE) is the major ACh hydrolyzing enzyme in plasma, and the aim of our study was to evaluate its association with low-grade systemic inflammation., Material and Methods: A total of 4,077 patients clinically managed in the Cardiology, Hypertension, and Digestive Medicine Units were included in our study. Three subclinical chronic inflammatory degrees were established in accordance with the high-sensitivity C-reactive protein (hsCRP) concentrations proposed, for low (< 1 mg/L), average (1-3 mg/L), and high (> 3-10 mg/L) cardiovascular disease risk estimation., Results: In male patients with subclinical chronic inflammation and hsCRP concentrations < 1 mg/L, a significant positive correlation was observed between BChE and hsCRP (p < 0.02); however, for hsCRP concentrations > 3 mg/L, the correlation between these variables in both sexes becomes significantly negative (p < 0.001), as in patients with acute inflammation (hsCRP > 10 mg/L). In all cases significant positive correlations were obtained between the BChE activities and albumin concentrations (p < 0.001)., Conclusions: The liver production of BChE and albumin occurs in a coupled fashion, and these biochemical variables may be considered as negative inflammatory reactants, whose serum levels are inversely associated with the increasing degree of subclinical inflammation.

Background: Valproic acid (VPA) apparent clearance (CL) estimated from total serum concentrations is analogous in elderly and non-elderly adult patients. As drug-protein binding decreases in old age, the aim of our study was to evaluate the confounding effect of the serum albumin concentration on the VPA apparent CL in elderly patients., Methods: In 102 epileptic out-patients treated with VPA in monotherapy, serum total steady-state trough concentrations (Css) were determined. Css concentrations were normalized for a 42 g/L albumin concentration (Css(N)), and the apparent CL and normalized apparent CL(N) were calculated., Results: A poor concordance of 53% was found in the classification of Css and Css(N) levels of VPA as subtherapeutic, therapeutic, or supratherapeutic dose. In the elderly (≥65 years) and non-elderly adult patients, the VPA apparent CL was similar; however, normalized apparent CL(N) was significantly lower in older patients (P < 0.01), with a 40% median decrease., Conclusions: Total VPA concentrations should be interpreted with caution, mainly in older patients, in which determination of unbound or normalized total drug concentrations may be clinically useful. Normalization of total concentrations permits an estimation of the masking effect of serum albumin concentrations on the VPA apparent CL in elderly patients.

Background: Patients treated with valproic acid (VPA) present a high incidence of non-alcoholic fatty liver disease (NAFLD) (around 61%). Several recent studies suggest that low copper stores could be associated with NAFLD, and a significant decrease of copper availability in VPA-treated patients has been described., Design and Methods: In 101 adult epileptic patients treated with valproic acid in monotherapy (n = 75) and polytherapy (n = 26) the copper availability was evaluated using the specific oxidase activity of ceruloplasmin (activity per unit mass of enzyme protein) and the copper/ceruloplasmin ratio. Copper deficiency was supposed in the cases in which this biochemical variable was smaller than the lower reference limit (333 U/g)., Results: The differences between the groups of patients with ceruloplasmin oxidase activity smaller or greater than 333 U/g for the serum levels of aminotransferases, gamma-glutamyltransferase, butyrylcholinesterase, cholesterol, triglycerides, and C-reactive protein, and the APRI and FIB-4 liver fibrosis scores were not statistically significant. Most patients (93%) had low APRI and FIB-4 scores, suggesting absence of significant liver fibrosis., Conclusions: The results obtained do not confirm the hypothesis of an association between diminished copper availability and NAFLD in patients treated with valproic acid.

Objectives: Gastrointestinal side-effects caused by mycophenolic acid (MPA) are frequent in liver transplant recipients, and in these cases a switch from two to three daily doses is usually recommended. However, a limited sampling strategy for the estimation of MPA area under the curve from 0 to 8 hours (AUC(0-8h)) has not been made., Design and Methods: In 22 liver transplant patients who were administered MPA three times daily, the trapezoidal extrapolated MPA AUC(0-8h) values using a sampling time from 0 to 2 hours were calculated., Results: A tentative therapeutic range for MPA AUC(0-8h) of about 20-40 μg.h/mL is proposed, and in the 13 patients with supratherapeutic values the total leukocyte blood count was significantly lower than in the 9 patients with AUC(0-8h) ≤ 40 μg.h/mL (P < 0.001). Significant negative correlations were found between the total leukocyte blood count and the MPA trough levels (r = -0.458; P < 0.05), AUC(0-8h) (r = -0.479; P < 0.05), and AUC(0-2h) (r = -0.437; P < 0.05). A significant correlation was found between the trapezoidal extrapolated AUC(0-8h) and trapezoidal AUC(0-2h) results (r = 0.850; P < 0.001)., Conclusions: The trapezoidal extrapolated AUC(0-8h), and possibly trapezoidal AUC(0-2h), may be useful for routine therapeutic MPA monitoring in liver transplant recipients in which the dosing frequency is increased from twice to three times a day.

Objectives: Therapeutic monitoring of sirolimus and everolimus is necessary in order to minimize adverse side-effects and to ensure effective immunosuppression. A sirolimus-dosing model using the concentration/dose ratio has been previously proposed for kidney transplant patients, and the aim of our study was the evaluation of this single model for the prediction of trough sirolimus and everolimus concentrations., Methods: Trough steady-state sirolimus concentrations were determined in several blood samples from each of 7 kidney and 9 liver maintenance transplant recipients, and everolimus concentrations from 20 kidney, 17 liver, and 3 kidney/liver maintenance transplant recipients. Predicted sirolimus and everolimus concentrations (Css), corresponding to the doses (D), were calculated using the measured concentrations (Css(0)) and corresponding doses (D(0)) on starting the study: Css = (Css(0))(D)/D(0)., Results: The diagnostic efficiency of the predicting model for the correct classification as subtherapeutic, therapeutic, and supratherapeutic values with respect to the experimentally obtained concentrations was 91.3% for sirolimus and 81.4% for everolimus in the kidney transplant patients. In the liver transplant patients the efficiency was 69.2% for sirolimus and 72.6% for everolimus, and in the kidney/liver transplant recipients the efficiency for everolimus was 67.9%., Conclusions: The model has an acceptable diagnostic efficiency (>80%) for the prediction of sirolimus and everolimus concentrations in kidney transplant recipients, but not in liver transplant recipients. However, considering the wide ranges found for the prediction error of sirolimus and everolimus concentrations, the clinical relevance of this dosing model is weak.

Background: Although high-performance liquid chromatography (HPLC) is the method of choice for blood sirolimus determination, the microparticle enzyme immunoassay (MEIA) run on the IMx analyser is widely used in therapeutic monitoring of this immunosuppressant agent. The aim of our study was to evaluate the possible determination of sirolimus using the fluorescence polarization immunoassay (FPIA) commercialized for everolimus quantification., Methods: Sirolimus concentrations were determined in whole-blood samples from liver and kidney transplant recipients using the Innofluor Certican FPIA (Seradyn Inc.) run on a TDx analyser (Abbott Laboratories), Sirolimus MEIA run on an IMx analyser (Abbott Laboratories), and HPLC (UV detection) methods., Results: The Innofluor FPIA has a similar cross-reactivity with everolimus and sirolimus, and the within- and between-run coefficients of variation obtained for sirolimus determination were 2.7%-13.3%. In analysing different blood samples from liver and kidney transplant patients the linear regressions obtained were: FPIA = 1.12 HPLC + 0.43 (n=104, r=0.874), MEIA = 1.14 HPLC (n=146, r=0.892), and FPIA = 1.00 MEIA + 0.29 (n=106, r=0.941). Better correlation coefficients were obtained between the methods in the liver transplant samples (r>or=0.900) than in the kidney transplant samples (r>or=0.849). No significant effect was found for sirolimus clearance or the blood hematocrit on the relationship between the results produced by both immunoassays and HPLC., Conclusion: The Innofluor FPIA is a valid alternative with an analogous performance to the MEIA for the therapeutic monitoring of sirolimus.

Objective: The aim of this study was to compare the estimated glomerular filtration rate (GFR) using the Cockcroft-Gault and the 4-, 5-, and 6-variable Modification of Diet in Renal Disease (MDRD) formulas for digoxin dose adjustment., Methods: Steady-state serum digoxin concentrations were determined in 100 patients with heart failure and normal to severely impaired renal function. Total clearance (CL) and predicted average concentrations of digoxin were calculated using general pharmacokinetic principles., Results: The mean+/-SEM (median) estimated GFR values were 48.9+/-2.8 (46.5) mL/min/1.73 m(2) using the Cockcroft-Gault formula, 61.4+/-3.6 (56.4) mL/min/1.73 m(2) using the MDRD4 formula, 56.8+/-3.3 (52.1) mL/min/1.73 m(2) using the MDRD5 formula, and 53.3+/-3.0 (48.7) mL/min/1.73 m(2) using the MDRD6 formula, with high correlation coefficients between the estimates (r > or = 0.928, P < 0.001). Significant correlations were found between the digoxin total CL and estimated GFR by the Cockcroft-Gault (r = 0.649, P < 0.001), MDRD4 (r = 0.634, P <0.001), MDRD5 (r = 0.635, P < 0.001), and MDRD6 (r = 0.652, P < 0.001) formulas. A significant negative correlation of the digoxin total CL/GFR ratio with estimated GFR was obtained (r = -0.356, P < 0.001), with a high variability for this ratio for GFR lower than 60 mL/min. Analogous correlation coefficients were found between the obtained and predicted digoxin concentrations calculated using the estimated GFR by the Cockcroft-Gault (r = 0.628, P < 0.001), MDRD4 (r = 0.642, P < 0.001), MDRD5 (r = 0.650, P < 0.001), and MDRD6 (r = 0.665, P < 0.001) formulas, with a wide dispersion between the values in all cases., Conclusion: For GFR lower than 60 mL/min, the high interindividual variation of the digoxin total CL found among patients with similar renal function is an important limiting factor in the prediction of digoxin dosage regimens.

Background: The proposed action mechanism and pharmacological activity of carbamazepine (CBZ) and its major metabolite, carbamazepine-10,11-epoxide (CBZE), are the same. The aim of our study was the investigation of the effect of concomitant antiepileptic treatment and renal insufficiency on the relative proportions of serum CBZ and CBZE., Methods: Serum trough steady-state CBZ and CBZE concentrations were determined by high-performance liquid chromatography (HPLC) in 140 epileptic patients treated with CBZ in monotherapy (n=100) and polytherapy with phenytoin, phenobarbital and valproate (n=40). The levels of CBZ were also determined using the Dade Behring enzyme multiplied immunoassay technique (EMIT). The glomerular filtration rate (GFR) was estimated from serum cystatin C using the Dade Behring nephelometric immunoassay., Results: The CBZE/CBZ and CBZ+CBZE/CBZEMIT ratios were significantly increased in 7 cases (3 in monotherapy and 4 in polytherapy) with GFR<60 mL/min/1.73m2 in relation to the patients treated in monotherapy or polytherapy having normal or mildly decreased renal function (p<0.001)., Conclusions: In patients with moderate to severe renal insufficiency the relative proportion of CBZE with respect to the parent drug is significantly increased. In these cases, the CBZ concentrations obtained using the EMIT, or other immunoassays having low CBZE cross-reactivity, may have an inadequate diagnostic efficiency.

Background: Published data on the performance of the new Dade Behring antibody conjugated magnetic immunoassay (ACMIA) for tacrolimus determination are scarce. The aim of this study was to compare the results obtained using the ACMIA and Abbott microparticle enzyme immunoassay (MEIA), which is the most widely used method for therapeutic tacrolimus monitoring., Methods: Trough tacrolimus concentrations were determined in 305 blood samples from kidney (n=138) and liver (n=167) transplant recipients using the ACMIA and MEIA immunoassays. The MEIA results were corrected for hematocrit values lesser than 30% and higher than 40% (Hermida et al. Clin Lab 2005; 51: 43-45)., Results: The obtained ACMIA within- and between-run variation coefficients (<10.8%) were acceptable. In the comparison between ACMIA and MEIA results in the blood samples studied, the regression equation was: ACMIA=1.02MEIA+0.29 (r=0.912, p<0.001), with an acceptable difference between the means (8.13+/-0.53 ng/mL vs. 7.62+/-0.50 ng/mL). However, in accordance with the well-established interference of the hematocrit on the MEIA results, a highly significant negative correlation between the MEIA/ACMIA ratio and the hematocrit values was obtained (r=-0.585, p<0.001). When the MEIA results were corrected according to the hematocrit (MEIAHtC), the regression with ACMIA levels was: ACMIA=1.08MEIAHtC-0.09 (r=0.926, p<0.001). This equation was analogous to that obtained between ACMIA and MEIA tacrolimus concentrations in the 164 blood samples with hematocrit of 30-40%., Conclusions: ACMIA is an acceptable option for therapeutic tacrolimus monitoring, with an important decrease in technician time in relation to the widely used MEIA.

Background: Carbamazepine (CBZ) occasionally causes haematological disorders such as thrombocytopenia, and recently a case of oxcarbazepine (OXCBZ)-induced thrombocytopenia has been described. The aim of our study was blood platelet count determination in epileptic patients treated with CBZ and OXCBZ, and its relationship with the dose and serum levels of these drugs and its metabolites., Methods: The serum levels of CBZ and its epoxide, and the pharmacologically active monohydroxy derivative of OXCBZ were determined in 137 patients treated with CBZ, and 60 patients treated with OXCBZ. The platelet count, mean platelet volume, and platelet size distribution width were also determined., Results: The difference between the platelet counts of the patient groups treated with CBZ and OXCBZ was not significant. No significant correlations between the platelet count and serum levels of the administered antiepileptic drugs and their metabolites were found. However, significant negative correlations between the platelet count and the daily doses of CBZ and OXCBZ were obtained (p<0.01). In 5 cases (4 treated with CBZ and 1 with OXCBZ) the platelet count was <150 x 10(9)/l., Conclusions: In accordance with the mean platelet volume and platelet distribution width, the thrombocytopenia observed in some of the patients studied was due to a hyper-destruction of peripheral blood platelets. However, the results obtained suggest that the mechanism of CBZ or OXCBZ-induced thrombocytopenia is not due to a direct toxicity of these drugs or their major metabolites on the circulating platelets. Although, the patients treated with OXCBZ shown a lower prevalence for thrombocytopenia (1.7%) than those treated with CBZ (2.9%), the routine platelet count monitoring in patients treated with both drugs may be recommended.

The debate continues regarding the possible interference of phenytoin metabolites in phenytoin immunoassays, and its clinical importance for patients with renal failure. The aim of this study was to compare the results obtained using the Abbott fluorescence polarization immunoassay (FPIA), Dade enzyme-multiplied immunoassay technique (EMIT), and high-performance liquid chromatography (HPLC) to establish the significance of the differences in conditions of renal failure. Thirty-six adult patients who had been treated with phenytoin and whose renal function ranged from normal to severely impaired were chosen for this study. In accordance with previously established validation criteria for analytical methods for the determination of drugs, a 15% bias from the HPLC phenytoin values was considered an acceptable limit. The mean (+/-SEM) glomerular filtration rate (GFR) of the patients was 37.5+/-4.6 mL/min (range = 10-102 mL/min). The mean values found using FPIA (10.8+/-1.2 microg/mL) and EMIT (10.8+/-1.3 microg/mL) presented acceptable deviations with respect to HPLC (10.5+/-1.2 microg/mL), and a high correlation was found among the results (N = 36) of the different methods (r > or = 0.987, P < 0.001). An FPIA deviation above the 15% bias limit with respect to HPLC was found only in two cases with very low serum phenytoin concentrations and low GFR values (< 20 mL/min), although it does not appear to be important in terms of adjusting drug dosage. According to our data, FPIA and EMIT gave accurate results for total phenytoin in serum samples from patients with renal failure., (2007 Wiley-Liss, Inc.)

Background: ss-N-acetylhexosaminidase (Hex) is a lysosomal hydrolase, whose determination in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) has provided discordant results., Methods: Total Hex and its isoenzymes Hex A and Hex B were determined using a thermodynamic procedure in the CSF of 27 patients with definitive MS, 8 with possible MS, 9 with meningitis, 14 with other neurological diseases, and in 10 controls without any neurological disease., Results: In the group of patients with definitive MS, the total Hex and Hex A were significantly higher than in the control group (p<0.001), with a possible association of greater enzymatic activities with the presence of oligoclonal bands and recent relapse; however, an overlap was detected for the activities of total Hex and its isoenzymes between the groups of patients with different neurological diseases. A significant correlation was obtained for neuron-specific enolase (NSE) with total Hex and Hex A and Hex B isoenzymes (p<0.001); however, in the partial correlation statistical significance was only obtained between NSE and Hex A (p<0.001) which is the most abundant Hex isoenzyme in the brain., Conclusions: Although the inflammatory process in MS mainly takes place in the perivascular zone, with little activity in the cerebral parenchyma, the significant increase of NSE and Hex A isoenzyme in CSF reveals a neuronal damage. The disease status may have effect on the CSF Hex activity.

The action of some anticonvulsant drugs as the causal agents of attacks of acute porphyria has been widely documented in the literature. However, little attention has been paid to the effect of these drugs on the urinary excretion of porphyrins in non-porphyric subjects. In a sample of 82 epileptic patients treated with phenobarbital (n = 54), phenytoin (n = 64), carbamazepine (n = 33), and valproate (n = 8), the daily doses were expressed according to a drug score that would reflect the capacity of these drugs as enzymatic inducers when administered in polytherapy. A significantly increased urinary excretion of D-glucaric acid (DGA) and porphyrins was found in this group of patients (P<0.001), with coproporphyrin being the major fraction in all cases (>60%). Urinary DGA had a highly significant correlation with the drug score (r = 0.783, P<0.001); however, no significant correlations were found between the urinary porphyrins and DGA (r = 0.005) or the drug score (r = 0.053). Neither was any significant relationship found between the urinary porphyrins and the serum activity of 5'-nucleotidase (r = 0.066) or the presence of a cholestasis objectivized through the presence of the isoform of gamma-glutamyltransferase with beta-globulins electrophoretic mobility. However, in a group of 10 patients a significant correlation was found between the urinary excretion of porphyrins and beta-N-acetylhexosaminidase (r = 0.790, P<0.01). Therefore, it does not appear that the liver enzyme induction, or even a subclinical cholestasis, produced by the antiepileptic drugs administered to these patients may serve to explain the increase in the urinary excretion of porphyrins. A possible renal origin is proposed for the increase of urinary porphyrins in these cases.

Background: The relationship between the progress of tubular damage and renal insufficiency in autosomal-dominant polycystic kidney disease (ADPKD) is a subject of doubtless interest, and is the object of this present work., Methods: A total of 92 adult ADPKD patients of both genders were studied, none of which presented end-stage renal disease (ESRD), and classified according to an ultrasound score based on kidney size and number of cysts. Urinary albumin and beta-N-acetylhexosaminidase (Hex) and its isoenzymes were determined, together with serum glutathione peroxidase, cystatin C, creatinine, and urea., Results: A frequent elevation of the urinary Hex was found and an alteration of its isoenzymatic profile, with 31% of the normotensive patients with normoalbuminuria already presenting an increased proportion of Hex B isoenzyme. Keeping age constant, a partial significant correlation was found between the ultrasound score and the proportion of Hex B (r = 0.352, P < 0.05), but not with albuminuria or cystatin C. In 42 patients the different biochemical variables were again determined after 1 year, finding that in the 13 normotensive patients with normoalbuminuria there had been a significant decrease in the concentration of cystatin C (P < 0.05), and a significant increase in the urinary excretion of albumin and Hex B isoenzyme (P < 0.05). By the other hand, in the other 29 patients with micro- or macroalbuminuria and hypertension, no significant differences were found., Conclusion: The results point toward an important participation of tubular damage in the pathogenesis of this disease. It may also be suggested that in normotensive and normoalbuminuric ADPKD patients, a gradual increase of glomerular filtration would be produced. After the start of hypertension and microalbuminuria, the glomerular filtration rate (GFR) would decrease progressively, although more slowly.

In patients with hypoalbuminemia, the total serum concentration of valproic acid may offer poor clinical information; however, very few clinical laboratories routinely analyze the free concentration of the drug. The aim of this study was to design a procedure to normalize the total concentration of valproic acid according to the level of serum albumin and using previously published free fraction values. In 121 adult patients, with albumin levels of 18 - 41 g/L, the total concentration of valproic acid was normalized using the derived equation: C(N) = alpha(H)C(H)/6.5, where alpha(H) is the free fraction of the drug corresponding to the patient's particular albuminemia and C(H) is the total concentration of valproic acid. The value of 6.5 corresponds to the free fraction of the drug for a serum albumin of 42 g/L (percentile 50 of the reference range). For total concentrations lower than 75 mg/L, the predicted normalized valproic acid concentrations were reasonably concordant with the observed normalized concentrations calculated using the data from a protein-binding study. In a significant number of cases, subtherapeutic concentrations of the drug became therapeutic and even supratherapeutic when corrected according to the albumin levels. Furthermore, cases with therapeutic drug concentrations frequently became supratherapeutic when normalized. The limitations and clinical applications of the proposed formula for normalizing the total concentration of valproic acid are presented. It is concluded that it may be useful for the posological management of hypoalbuminemic patients when the free concentration of the drug is not available, and decisions have to be made based on the total serum concentration.

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.

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.

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.

The activation energy (Ea) of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52) was determined with 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide as substrate, with a much higher value being found for the Hex B isoenzyme (Ea = 75.1 kJ/mol) than for the Hex A isoenzyme (Ea = 41.8 kJ/mol). This fact allowed for the development of a fast and reliable thermodynamic method to determine the isoenzyme composition of Hex in different biological specimens (serum/plasma, saliva, cerebrospinal fluid, seminal plasma, urine, and leukocyte lysates). The results in serum given by the proposed method may be superimposed upon those obtained by the heat inactivation assay of O'Brien et al. (N Engl J Med 1970;273:15-20), and the catalytic activity calculated for Hex A offers a good correlation with that obtained by using the specific substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6 sulfate (n = 25, r = 0.953).