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1.
Ther Drug Monit ; 45(2): 173-190, 2023 04 01.
Article in English | MEDLINE | ID: mdl-36920502

ABSTRACT

ABSTRACT: Immunosuppressants have a narrow therapeutic index (NTIDs). Indisputably cyclosporine, tacrolimus, everolimus, and sirolimus have NTIDs, and only in the case of mycophenolic acid, a scientific discussion has not been yet concluded. Their specificities highlight the implications for generics introduced into the drug market, more precisely, with bioequivalence testing. In the European Union, the European Medicines Agency (EMA) released the "Guideline on the Investigation of Bioequivalence." The bioequivalence (BE) of the generic (tested, T) versus original (reference, R) product should be confirmed by obtaining a 90% confidence interval (CI) for the T:R ratio of each of the 2 decisive pharmacokinetic parameters, namely, the area under the curve (AUC) between 90.00% and 111.11%. A similar approach (90.00%-112.00%) for AUC was adopted by the Canadian Agency for Drugs and Technologies in Health (CADTH) for NTIDs; however, the US Food and Drug Administration is still based on classic acceptance criteria: 90% CI between 80.00% and 125.00% but with special requirements of BE testing. A discussion about long-expected global consensus was performed in this study based on the literature concerning BE testing in the case of NTIDs. The narrow acceptance criteria reduce the potential mean difference in bioavailability between generic and original products by a few percent. To identify this problem, special attention has been paid to switching drugs (generic-generic, original-generic) and therapeutic drug monitoring after conversion (TDM). There is no global consensus on the acceptance criteria for the BE of generic drugs; therefore, consensus and harmonization are strictly necessary. This study presents a review of the generic drug market and its classification by manufacturers, drug agencies, and dates of marketing authorization. Guidelines for TDM optimization (during switching/conversion) have been proposed. Physicians and clinical pharmacists should pay special attention to switching immunosuppressive drugs between original versus generic formulations, and generic versus generic formulations. Patients and their families should be educated on the risks associated with uncontrolled conversion.


Subject(s)
Drug Monitoring , Drugs, Generic , Humans , Drugs, Generic/therapeutic use , Drugs, Generic/pharmacokinetics , Canada , Immunosuppressive Agents/therapeutic use , Immunosuppressive Agents/pharmacokinetics , Therapeutic Equivalency , Pharmaceutical Preparations , Immunosuppression Therapy
2.
Molecules ; 26(23)2021 Nov 29.
Article in English | MEDLINE | ID: mdl-34885834

ABSTRACT

The aim of the work was to prepare a simple but reliable HPLC-UV method for the routine monitoring of mycophenolic acid (MPA). Sample preparation was based on plasma protein precipitation with acetonitrile. The isocratic separation of MPA and internal standard (IS) fenbufen was made on Supelcosil LC-CN column (150 × 4.6 mm, 5 µm) using a mobile phase: CH3CN:H2O:0.5M KH2PO4:H3PO4 (260:700:40:0.4, v/v). UV detection was set at 305 nm. The calibration covered the MPA concentration range: 0.1-40 µg/mL. The precision was satisfactory with RSD of 0.97-7.06% for intra-assay and of 1.92-5.15% for inter-assay. The inaccuracy was found between -5.72% and +2.96% (+15.40% at LLOQ) and between -8.82% and +5.31% (+19.00% at LLOQ) for intra- and inter-assay, respectively, fulfilling acceptance criteria. After a two-year period of successful application, the presented method has been retrospectively calibrated using the raw data disregarding the IS in the calculations. The validation and stability parameters were similar for both calculation methods. MPA concentrations were recalculated and compared in 1187 consecutive routine therapeutic drug monitoring (TDM) trough plasma samples from mycophenolate-treated patients. A high agreement (r2 = 0.9931, p < 0.0001) of the results was found. A Bland-Altman test revealed a mean bias of -0.011 µg/mL (95% CI: -0.017; -0.005) comprising -0.14% (95% Cl: -0.39; +0.11), whereas the Passing-Bablok regression was y = 0.986x + 0.014. The presented method can be recommended as an attractive analytical tool for medical (hospital) laboratories equipped with solely basic HPLC apparatus. The procedure can be further simplified by disapplying an internal standard while maintaining appropriate precision and accuracy of measurements.


Subject(s)
Chromatography, High Pressure Liquid/standards , Mycophenolic Acid/blood , Calibration , Humans , Limit of Detection , Reference Standards , Reproducibility of Results
3.
Ther Drug Monit ; 43(2): 150-200, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33711005

ABSTRACT

ABSTRACT: When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.


Subject(s)
Drug Monitoring , Immunosuppressive Agents/administration & dosage , Mycophenolic Acid/administration & dosage , Organ Transplantation , Area Under Curve , Consensus , Graft Rejection/prevention & control , Humans
4.
Ther Drug Monit ; 41(3): 261-307, 2019 06.
Article in English | MEDLINE | ID: mdl-31045868

ABSTRACT

Ten years ago, a consensus report on the optimization of tacrolimus was published in this journal. In 2017, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicity (IATDMCT) decided to issue an updated consensus report considering the most relevant advances in tacrolimus pharmacokinetics (PK), pharmacogenetics (PG), pharmacodynamics, and immunologic biomarkers, with the aim to provide analytical and drug-exposure recommendations to assist TDM professionals and clinicians to individualize tacrolimus TDM and treatment. The consensus is based on in-depth literature searches regarding each topic that is addressed in this document. Thirty-seven international experts in the field of TDM of tacrolimus as well as its PG and biomarkers contributed to the drafting of sections most relevant for their expertise. Whenever applicable, the quality of evidence and the strength of recommendations were graded according to a published grading guide. After iterated editing, the final version of the complete document was approved by all authors. For each category of solid organ and stem cell transplantation, the current state of PK monitoring is discussed and the specific targets of tacrolimus trough concentrations (predose sample C0) are presented for subgroups of patients along with the grading of these recommendations. In addition, tacrolimus area under the concentration-time curve determination is proposed as the best TDM option early after transplantation, at the time of immunosuppression minimization, for special populations, and specific clinical situations. For indications other than transplantation, the potentially effective tacrolimus concentrations in systemic treatment are discussed without formal grading. The importance of consistency, calibration, proficiency testing, and the requirement for standardization and need for traceability and reference materials is highlighted. The status for alternative approaches for tacrolimus TDM is presented including dried blood spots, volumetric absorptive microsampling, and the development of intracellular measurements of tacrolimus. The association between CYP3A5 genotype and tacrolimus dose requirement is consistent (Grading A I). So far, pharmacodynamic and immunologic biomarkers have not entered routine monitoring, but determination of residual nuclear factor of activated T cells-regulated gene expression supports the identification of renal transplant recipients at risk of rejection, infections, and malignancy (B II). In addition, monitoring intracellular T-cell IFN-g production can help to identify kidney and liver transplant recipients at high risk of acute rejection (B II) and select good candidates for immunosuppression minimization (B II). Although cell-free DNA seems a promising biomarker of acute donor injury and to assess the minimally effective C0 of tacrolimus, multicenter prospective interventional studies are required to better evaluate its clinical utility in solid organ transplantation. Population PK models including CYP3A5 and CYP3A4 genotypes will be considered to guide initial tacrolimus dosing. Future studies should investigate the clinical benefit of time-to-event models to better evaluate biomarkers as predictive of personal response, the risk of rejection, and graft outcome. The Expert Committee concludes that considerable advances in the different fields of tacrolimus monitoring have been achieved during this last decade. Continued efforts should focus on the opportunities to implement in clinical routine the combination of new standardized PK approaches with PG, and valid biomarkers to further personalize tacrolimus therapy and to improve long-term outcomes for treated patients.


Subject(s)
Immunosuppressive Agents/therapeutic use , Tacrolimus/therapeutic use , Consensus , Drug Monitoring/methods , Genotype , Graft Rejection/genetics , Graft Rejection/prevention & control , Humans , Organ Transplantation/methods , Precision Medicine/methods
6.
Eur J Clin Pharmacol ; 75(3): 371-379, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30430214

ABSTRACT

PURPOSE: To evaluate the relationship between total and free MPA pharmacokinetic (PK) parameters and renal outcome markers, and to verify whether conducting therapeutic drug monitoring (TDM) in lupus nephritis (LN) patients would be of value in routine clinical practice. METHODS: Eighty-four samples were collected from sixteen LN patients. Total and free MPA concentrations were measured at predose, 0.5 and 2 h after mycophenolate mofetil (MMF) intake. Area under the concentration time curve from 0 to 2 h (AUC0-2) and free fraction were calculated. RESULTS: High between-patient variability was observed (CV% of 53.5% for dose-normalized total MPA AUC0-2). A significant but weak correlation between dose-normalized total C0 and AUC0-2 was noted (r = 0.5699). Dose-normalized total C0 above 2.76 µg/mL·g may indicate patients with eGFR < 81 mL/min with sensitivity of 83.3% and specificity of 75.0%. Hypoalbuminemic LN patients demonstrated significantly elevated MPA free fraction when compared with patients with serum albumin concentration ≥ 3.5 g/dL (1.49 ± 0.64% vs 1.08 ± 0.75%). CONCLUSION: This study examined relationship between free and total pharmacokinetic MPA parameters as well as the effect of hypoalbuminemia on MPA plasma protein binding in adult LN patients. The study results suggest that TDM of MPA in LN seems to be a more reasonable approach than the fixed-dose protocol. Moreover, predose total MPA concentration may be a possible estimation of MPA exposure, while monitoring free rather than total MPA may be more beneficial in hypoalbuminemic patients.


Subject(s)
Drug Monitoring , Immunosuppressive Agents/blood , Kidney/drug effects , Lupus Nephritis/drug therapy , Mycophenolic Acid/blood , Adult , Area Under Curve , Dose-Response Relationship, Drug , Female , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/therapeutic use , Kidney/metabolism , Kidney Function Tests , Lupus Nephritis/blood , Male , Mycophenolic Acid/administration & dosage , Mycophenolic Acid/therapeutic use
7.
Article in English | MEDLINE | ID: mdl-30530114

ABSTRACT

The aim of this work was to develop and to validate LC-MS/MS method for tacrolimus (TAC) determination in whole blood samples using two different types of internal standards (IS): an isotope-labeled TAC13C,D2 and a structural analog ascomycin (ASC). Matrix effects (ME) were evaluated to determine their influence on validation parameters. The LC-MS/MS analyses were performed using a 4000 QTRAP® mass spectrometer (AB Sciex) coupled to a HPLC 1260 Infinity system (Agilent Technologies). The [M + NH4]+ adducts were monitored with mass transitions of: 821.5 → 768.4 m/z for TAC, 809.5 → 756.4 m/z for ASC, and 824.6 → 771.5 m/z for TAC13C,D2. Blood samples were treated with 0.1 mol/L zinc sulfate - acetonitrile (50:50, v/v) then extracted with tert-butyl methyl ether. ME evaluations were performed by preextraction addition (n = 6), postextraction addition (n = 8), and repeated measures (n = 8) of reference solutions, separately for both ISs. ME, absolute recovery (AR) and process efficiency (PE) were calculated. Low (1.5 ng/mL) and high (16 ng/mL) TAC concentrations were tested. The method was successfully calibrated in a range of: 0.5-20 ng/mL. Both ISs provided satisfactory imprecision (<3.09% and <3.63% for TAC13C,D2 and ASC, respectively) and accuracy (99.55-100.63% and 97.35-101.71% for TAC13C,D2 and ASC, respectively). Similar ARs were found for all three compounds yielding: 74.89-76.36% for TAC, 78.37% for TAC13C,D2 and 75.66% for ASC. Significant ME were observed yielding on the average: -16.04% and -29.07% for TAC, -16.64% for TAC13C,D2 and -28.41% for ASC. Consequently, PE was 64.11% and 53.12% for TAC, 65.35% for TAC13C,D2 and 54.18% for ASC. ME for TAC were perfectly compensated (TAC/IS ratio) in each sample resulting in mean percent value of: 0.89% and -0.97% for TAC13C,D2 and ASC, respectively. An IS ascomycin presented a performance equivalent to TAC13C,D2 in LC-MS/MS method developed for TAC monitoring.


Subject(s)
Chromatography, Liquid/methods , Tacrolimus/blood , Tandem Mass Spectrometry/methods , Humans , Limit of Detection , Linear Models , Reference Standards , Reproducibility of Results
8.
Biomed Chromatogr ; 31(10)2017 Oct.
Article in English | MEDLINE | ID: mdl-28317135

ABSTRACT

The aim of this study was to develop and validate fully the liquid chromatography-tandem mass spectrometry method for free mycophenolic acid (MPA) concentration measurements in plasma ultrafiltrate that will be reliable and simple in preparation with deuterated MPA (MPA-d3) chosen as an internal standard. The chromatographic separation was made with Zorbax Eclipse XDB-C18 column (4.6 × 150 mm) using a gradient of two solutions as a mobile phase: (A) water and (B) methanol, each containing 0.1% formic acid and 2.5 mm ammonium acetate. Satisfactory repeatability of retention times was achieved with average values of 7.54 ± 0.20 min and 7.50 ± 0.19 min for MPA and MPA-d3, respectively. The method was selective, with no carry-over or matrix effect observed. The analytical range was proven for MPA ultrafiltrate concentrations of 1-500 ng/mL. The accuracy and precision fell within the acceptance criteria for intraday (accuracy: 100.63-110.46%, imprecision: 6.23-7.76%), as well as interday assay (accuracy: 98.81-110.63%; imprecision: 5.36-10.22%). The method was used for free MPA determination in plasma samples from patients treated with mycophenolate mofetil. To the best of our knowledge this is the first liquid chromatography-tandem mass spectrometry method for free MPA monitoring using MPA-d3 that allows to measure plasma ultrafiltrate concentrations as low as 1 ng/mL.


Subject(s)
Chromatography, Liquid/methods , Mycophenolic Acid/blood , Tandem Mass Spectrometry/methods , Drug Stability , Humans , Limit of Detection , Linear Models , Mycophenolic Acid/chemistry , Reproducibility of Results , Ultrafiltration
9.
Ther Drug Monit ; 38(2): 143-69, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26982492

ABSTRACT

In 2014, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology called a meeting of international experts to provide recommendations to guide therapeutic drug monitoring (TDM) of everolimus (EVR) and its optimal use in clinical practice. EVR is a potent inhibitor of the mammalian target of rapamycin, approved for the prevention of organ transplant rejection and for the treatment of various types of cancer and tuberous sclerosis complex. EVR fulfills the prerequisites for TDM, having a narrow therapeutic range, high interindividual pharmacokinetic variability, and established drug exposure-response relationships. EVR trough concentrations (C0) demonstrate a good relationship with overall exposure, providing a simple and reliable index for TDM. Whole-blood samples should be used for measurement of EVR C0, and sampling times should be standardized to occur within 1 hour before the next dose, which should be taken at the same time everyday and preferably without food. In transplantation settings, EVR should be generally targeted to a C0 of 3-8 ng/mL when used in combination with other immunosuppressive drugs (calcineurin inhibitors and glucocorticoids); in calcineurin inhibitor-free regimens, the EVR target C0 range should be 6-10 ng/mL. Further studies are required to determine the clinical utility of TDM in nontransplantation settings. The choice of analytical method and differences between methods should be carefully considered when determining EVR concentrations, and when comparing and interpreting clinical trial outcomes. At present, a fully validated liquid chromatography tandem mass spectrometry assay is the preferred method for determination of EVR C0, with a lower limit of quantification close to 1 ng/mL. Use of certified commercially available whole-blood calibrators to avoid calibration bias and participation in external proficiency-testing programs to allow continuous cross-validation and proof of analytical quality are highly recommended. Development of alternative assays to facilitate on-site measurement of EVR C0 is encouraged.


Subject(s)
Drug Monitoring , Everolimus/pharmacokinetics , Everolimus/therapeutic use , Calcineurin Inhibitors/pharmacokinetics , Calcineurin Inhibitors/therapeutic use , Calibration , Consensus , Glucocorticoids/pharmacokinetics , Glucocorticoids/therapeutic use , Humans , Immunosuppressive Agents/pharmacokinetics , Immunosuppressive Agents/therapeutic use
10.
Ther Drug Monit ; 38(2): 170-89, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26982493

ABSTRACT

Monitoring immunosuppressive drugs (ISDs) in blood or plasma is still a key therapeutic drug monitoring (TDM) application in clinical settings. Narrow target ranges and severe side effects at drug underexposure or overexposure make accurate and precise measurements a must. This overview prepared by the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology is intended to serve as a summary and guidance document describing the current state-of-the-art in the TDM of ISDs.


Subject(s)
Drug Monitoring , Immunosuppressive Agents/blood , Immunosuppressive Agents/pharmacokinetics , Humans
11.
Article in English | MEDLINE | ID: mdl-26310898

ABSTRACT

BACKGROUND: Measurements of plasma concentrations of free normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MTY) constitute the most diagnostically accurate screening test for pheochromocytomas and paragangliomas. The aim of this article is to present the results from a validation of an analytical method utilizing high performance liquid chromatography with coulometric detection (HPLC-CD) for quantifying plasma free NMN, MN and MTY. Additionally, peak integration by height and area and the use of one calibration curve for all batches or individual calibration curve for each batch of samples was explored as to determine the optimal approach with regard to accuracy and precision. METHODS: The method was validated using charcoal stripped plasma spiked with solutions of NMN, MN, MTY and internal standard (4-hydroxy-3-methoxybenzylamine) with the exception of selectivity which was evaluated by analysis of real plasma samples. Calibration curve performance, accuracy, precision and recovery were determined following both peak-area and peak-height measurements and the obtained results were compared. The most accurate and precise method of calibration was evaluated by analyzing quality control samples at three concentration levels in 30 analytical runs. RESULTS: The detector response was linear over the entire tested concentration range from 10 to 2000pg/mL with R(2)≥0.9988. The LLOQ was 10pg/mL for each analyte of interest. To improve accuracy for measurements at low concentrations, a weighted (1/amount) linear regression model was employed, which resulted in inaccuracies of -2.48 to 9.78% and 0.22 to 7.81% following peak-area and peak-height integration, respectively. The imprecisions ranged from 1.07 to 15.45% and from 0.70 to 11.65% for peak-area and peak-height measurements, respectively. The optimal approach to calibration was the one utilizing an individual calibration curve for each batch of samples and peak-height measurements. It was characterized by inaccuracies ranging from -3.39 to +3.27% and imprecisions from 2.17 to 13.57%. CONCLUSIONS: The established HPLC-CD method enables accurate and precise measurements of plasma free NMN, MN and MTY with reasonable selectivity. Preparing calibration curve based on peak-height measurements for each batch of samples yields optimal accuracy and precision.


Subject(s)
Chromatography, High Pressure Liquid/methods , Dopamine/analogs & derivatives , Electrochemical Techniques/methods , Metanephrine/blood , Normetanephrine/blood , Dopamine/blood , Humans , Reproducibility of Results
12.
Ther Drug Monit ; 37(3): 311-8, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25380305

ABSTRACT

BACKGROUND: The aim of this study was to evaluate particle enhanced turbidimetric inhibition immunoassay (PETINIA) recently developed for mycophenolic acid (MPA) determination in plasma and to compare it with a reference high-performance liquid chromatography (HPLC) method, using samples from heart transplant recipients. The results are presented in the context of PETINIA being compared with enzyme multiplied immunoassay technique (EMIT). METHODS: PETINIA evaluation was performed using 194 routine trough plasma samples at steady state. EMIT was evaluated using 677 samples from 61 steady-state 12-hour profiles obtained from 35 heart transplant patients. Evaluation was undertaken on a Dimension EXL 200 analyzer (PETINIA) and on a Viva-E analyzer (EMIT). RESULTS: The mean MPA concentration measured by PETINIA was significantly higher than that measured by high-performance liquid chromatography combined with UV detector (2.36 ± 1.30 mcg/mL versus 1.82 ± 1.23 mcg/mL, respectively, P < 0.0001). Bland-Altman analysis revealed a mean bias of 0.54 mcg/mL [95% confidence interval (CI), 0.49-0.59] comprising 33.48% (95% CI, 30.34-36.61). Passing-Bablok regression was: y = 1.100x + 0.38 (95% CI for slope: 1.044-1.154 and for intercept: 0.30-0.47). Regardless of a significant observed correlation (r = 0.9230, P < 0.0001), the statistical analyses showed a significant difference between PETINIA and the reference chromatographic method. The mean MPA concentration measured by EMIT was significantly higher than that measured by HPLC (7.48 ± 8.34 mcg/mL versus 5.57 ± 6.61 mcg/mL, respectively, P < 0.0001) with a mean bias of 1.91 mcg/mL (95% CI, 1.75-2.07) comprising 35.91% (95% CI, 34.37-37.45). The significant difference between EMIT and HPLC was confirmed by Passing-Bablok regression: y = 1.300x + 0.24 (95% CI for slope: 1.279-1.324 and for intercept: 0.18-0.29). The analysis of the determinations, grouped by sampling time, revealed positive bias between EMIT and HPLC ranging from 24.54% to 42.77% and inversely proportional to MPA concentrations with r = 0.9122 (P < 0.001). CONCLUSIONS: The new immunochemical PETINIA method was associated with significantly higher MPA concentrations in routine therapeutic drug monitoring samples from heart transplant patients. The magnitude of the MPA overestimation was similar to that observed by use of the EMIT method.


Subject(s)
Drug Monitoring/methods , Enzyme Multiplied Immunoassay Technique , Heart Transplantation , Mycophenolic Acid/blood , Nephelometry and Turbidimetry/methods , Adolescent , Adult , Aged , Chromatography, High Pressure Liquid , Female , Humans , Immunosuppressive Agents/blood , Male , Middle Aged , Spectrophotometry, Ultraviolet , Young Adult
13.
Article in English | MEDLINE | ID: mdl-23217316

ABSTRACT

The paper presents an HPLC method for cefazolin determination in human serum. The preparation step was based on serum protein precipitation with acetonitrile followed by supernatant evaporation and sample reconstitution in water before injection. The separation of cefazolin and internal standard cefamandole was performed at ambient temperature under isocratic conditions on LiChrosorb RP8-5 column (250mm×4.6mm) using the mixture: CH(3)CN:H(2)O:0.5M KH(2)PO(4) (100:894:6, v/v) as a mobile phase with a flow rate of 1.5mL/min. UV detection was performed at 272nm with LLOQ of 0.2µg/mL. The precision was satisfactory in the whole range tested with RSD of 2.3-12.5% (accuracy: from -2.3% to +3.6%) and of 1.7-7.1% (accuracy: from -3.5% to +1.1%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze-thaw and short- and long-term stability testing. Finally, the procedure described was found resistant to potential human errors.


Subject(s)
Cefazolin/blood , Chromatography, High Pressure Liquid/methods , Cefazolin/chemistry , Chromatography, High Pressure Liquid/standards , Drug Stability , Humans , Linear Models , Reproducibility of Results , Sensitivity and Specificity , Temperature
15.
Folia Neuropathol ; 41(2): 65-8, 2003.
Article in English | MEDLINE | ID: mdl-12899198

ABSTRACT

In patients with dementia, 29 diagnosed as probably suffering from Alzheimer's disease and 46 subjects with dementia of vascular origin, and in 41 non demented control subjects LDL oxidation in vitro was compared in carriers of various apolipoprotein E alleles. Restriction isotyping was performed by gene amplification and cleavage with Hhal, LDL oxidation was investigated by determination of conjugated dienes and vitamin E (alpha tocopherol) plasma level was measured by HPLC. In subjects with dementia oxidation of LDL was shown to be higher in carriers of epsilon4 allele as compared with non-carriers of this allele. It was especially observed in the propagation phase, which illustrates oxidation intensity after the exhaustion of the antioxidant reserve in LDL. Vitamin E level did not show differences between carriers of different alleles. It is concluded that the differences in oxidation susceptibility of LDL between demented subjects possessing particular apolipoprotein E forms can result partially from differing antioxidant properties of apolipoprotein E isoforms and, in a substantial degree, from the size and quality of LDL.


Subject(s)
Apolipoproteins E/genetics , Dementia/genetics , Dementia/metabolism , Lipoproteins, LDL/metabolism , Polymorphism, Genetic/genetics , Aged , Aged, 80 and over , Apolipoprotein E4 , Female , Heterozygote , Humans , Male , Middle Aged , Oxidation-Reduction , Statistics, Nonparametric
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