Effect of postpartum iron supplementation on red cell and iron parameters in non-anaemic iron-deficient women: a randomised placebo-controlled study.

OBJECTIVE: To investigate the effect of oral iron on postpartum red cell and iron parameters in non-anaemic women with iron deficiency. DESIGN: Randomised study of supplementation with oral iron sulphate 80 mg daily or placebo for 12 weeks starting 24-48 hours after delivery, with visits antepartum and 1, 4, 6 and 12 weeks postpartum. SETTING: Swiss university hospital obstetric unit. PARTICIPANTS: Fifty-two women with antenatal iron deficiency (serum ferritin <15 microg/L) and no antenatal or postnatal anaemia (haemoglobin >11 g/dL up to 48 hours before delivery, and >10 g/dL postpartum), divided into two groups comparable in antenatal iron status. METHODS: Supplementation was started 24-48 hours after delivery (visit 1:V1). Additional tablets were issued one week after V1 (V2), four weeks after V1 (V3) and six weeks after V1 (V4). The last visit took place 12 weeks after visit 1 and 6 weeks after visit 4 (V5). Patients were required to return blisters and boxes whether they were used and unused at each visit and compliance was assessed by counting the tablets. Blood samples for haematology and iron status testing were taken before delivery and at each visit. MAIN OUTCOME MEASURES: Iron status (serum ferritin, hypochromic red cells, iron, transferrin saturation, soluble transferrin receptor concentration); erythropoiesis (standard parameters, including reticulocyte indices); and inflammatory response (serum neopterin, C-reactive protein, white cell count) in five-datapoint profiles. RESULTS: Increased ferritin (P= 0.0004) and transferrin saturation (P= 0.03), decreased soluble transferrin receptors (P= 0.02); increased haemoglobin (P= 0.02) and decreased hypochromic red cells (P= 0.04) compared with placebo at 12 weeks, with no differences in other red cell or reticulocyte parameters. There was a positive correlation between C-reactive protein and postpartum ferritin. No correlation was observed in the puerperium between C-reactive protein and hypochromic red cells or soluble transferrin receptors. CONCLUSIONS: Haemoglobin levels and iron stores in women with term gestational iron deficiency benefit significantly from iron supplementation compared with placebo, even in an industrialised population.

CCTTT-repeat polymorphism of the inducible nitric oxide synthase is not associated with HIV pathogenesis.

Nitric oxide (NO) produced by the inducible form of nitric oxide synthase (iNOS) has bactericidal and virocidal effects. Although NO synthesis and iNOS expression in macrophages affect several aspects of human immunodeficiency virus (HIV) type-1 pathogenesis, their role in HIV disease remains largely unknown. In humans, the expression of iNOS is influenced by a functional CCTTT-repeat polymorphism in the promoter region of the gene. We investigated the association of this polymorphism with HIV pathogenesis in naive HIV-infected patients before the initiation of antiretroviral therapy. The allele frequencies of the iNOS CCTTT-repeat polymorphism were assessed by PCR in 857 patients from the Swiss HIV Cohort Study, including rapid progressors and long-term nonprogressors, and in 240 healthy volunteers. In HIV-infected patients, the initial viral load and the decline in total CD4 cells was calculated to estimate disease progression. Allele frequencies of the iNOS CCTTT-repeat polymorphism were similar between the HIV-infected and noninfected blood donors. In treatment-naive HIV-positive patients, there was no association of the iNOS polymorphism with viral load or with the course of CD4 cells. Regulation of iNOS expression by the functional CCTTT-polymorphism does not modify HIV pathogenesis.

Factor V Leiden mutation in postthrombotic and non-postthrombotic venous ulcers.

OBJECTIVE: To determine the prevalence of the factor V Leiden mutation in patients with postthrombotic and non-postthrombotic venous ulcers. DESIGN: Case-control study. SETTING: Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland. PARTICIPANTS: Seventy-three consecutive outpatients and inpatients with venous ulcers and 45 age- and sex-matched control subjects (matched to the 42 patients with postthrombotic syndrome). MAIN OUTCOME MEASURES: Frequency of postthrombotic and non-postthrombotic findings in patients with venous ulcers. Prevalence of the factor V Leiden mutation in these different subgroups. RESULTS: Postthrombotic syndrome was identified as the cause of 42 (58%; 95% confidence interval [CI], 45%-69%) of 73 venous ulcers, and the remainder were caused by primary valvular insufficiency. In postthrombotic ulcers, the prevalence of the factor V Leiden mutation was 38% (95% CI, 24%-54%) (16/42), which corresponds to an odds ratio of 13.2 (95% CI, 2.8-62.3; P<.001). In non-postthrombotic venous ulcers, the prevalence was 16% (95% CI, 5%-34%) (5/31), which corresponds to an odds ratio of 3.2 (95% CI, 1.0-10.0; P =.07). CONCLUSIONS: The factor V Leiden mutation is highly prevalent in patients with postthrombotic venous ulcers. Even patients with non-postthrombotic venous ulcers show a moderately elevated prevalence of the factor V Leiden mutation. Some of the latter might be misclassified because of near-to-perfect revascularization after asymptomatic deep venous thrombosis. However, as long as the therapeutic consequences of the factor V Leiden mutation are not established, systematic screening cannot be recommended in patients with venous ulcers.

Effect of alcohol on postprandial lipemia with and without preprandial exercise.

OBJECTIVE: Different factors such as exercise habits and alcohol consumption may modulate postprandial lipid metabolism. What are the effects of alcohol on postprandial metabolism in untrained and trained individuals? METHODS: The postprandial lipid response to an oral fat load (1 g fat per kg body weight (bw)) with and without alcohol (0.5 g/kg bw) was evaluated in physically trained healthy young men (T, n = 12, mean +/- SD age 27 +/- 3 years. BMI 21.6 +/- 1.4 kg/m2) after a premeal running session and in untrained healthy young men (UT, n = 8, age 24 +/- 1 years, BMI 23.2 +/- 1.8 kg/m2) without a premeal exercise session. The T subjects ingested 35.5 +/- 2.7 g alcohol, the UT subjects 38 +/- 0.6 g. Fat was given as butter and the carbohydrates as marmalade and zwieback (rusk). The T subjects received 1.20 +/- 0.05 g fat and 1.02 +/- 0.04 g carbohydrates per kilogram lean body mass. The corresponding numbers for the UT subjects were 1.28 +/- 0.08 g and 1.20 +/- 0.06 g. The postprandial lipemia was observed for an eight-hour period. RESULTS: Alcohol led to an increase to the triacylglycerol area under the curve (AUC) in the T subjects from 7.4 +/- 0.4 mmol/L * h on the control day to 11.3 +/- 0.9 mmol/L * h (p = 0.001). The corresponding numbers in the UT subjects were 13.4 +/- 2.3 mmol/L * h to 19.4 +/- 3.5 mmol/L * h (p = 0.004). Alcohol intake and physical activity training were the major determinants of the triacylglycerol (TG) AUC in these subjects. CONCLUSION: The ingestion of a high fat meal in combination with alcohol leads to an increased in the postprandial lipemia independently from the level of training. It is suggested that this unfavorable effect of alcohol and a high fat diet could be modified by fat restriction or a combination of a premeal exercise session and a higher level of physical activity training.

Alkaline phosphatase activity: new assay for the Reflotron system. Results of the evaluation in eight clinical laboratories.

A new reagent carrier, Reflotron ALP, has been developed for the Reflotron system, allowing easy and rapid measurement (in less than 3 minutes) of alkaline phosphatase (ALP) activity in capillary blood, venous blood, heparinized plasma or serum. The evaluation of the analytical performance of the assay was carried out at eight clinical laboratories. The study of the imprecision using the measurements in human samples resulted in coefficients of variation ranging from 1.3% to 4.6% (within-run) and from 3.2% to 4.0% (day-to-day). The analytical specificity of the Reflotron ALP assay agrees well with ALP methods using a N-methyl-D-glucamine buffer solution. The calibration of the Reflotron ALP assay, however, is related to the reference intervals for ALP methods using a diethanolamine buffer solution. Method comparisons were performed with the ALP method on Hitachi instruments using diethanolamine buffer. Reflotron ALP measurements in blood and plasma in 157 randomly selected split samples showed excellent agreement (slope: 0.99; intercept: 0.7 U/l; median bias: 2.3%; median difference from the comparison method: -0.3%). Specimens from pregnant women and adolescents were excluded from this study. Differing values were obtained in a method comparison using 48 samples containing predominantly the ALP bone isoform (slope: 0.81; intercept: 31.5 U/l; median bias: 5.7%; median difference from the comparison method: -12.2%). Regression analysis of the results from 21 sera with prevailing placental ALP gave a slope of 1.51, and an intercept of -41.1 U/l (median bias: 8.6%; median difference from the comparison method: 35.6%). Reflotron ALP was compared with three different wet chemistry procedures using different buffer compounds: N-methyl-D-glucamine or diethanolamine or 2-amino-2-methyl-1-propanol. In samples containing predominantly ALP isoforms not of liver origin, the measurements with N-methyl-D-glucamine buffer gave the best fit with respect to Reflotron. In an interference study with 18 drugs, no effect on the test results could be detected. Total bilirubin up to 750 micromol/l and hemolysis up to 1.7 g/l free hemoglobin did not influence the test. Reflotron ALP proved to be an easy and rapid method with excellent precision. The accuracy related to an ALP method using diethanolamine buffer was good. The systematic differences for ALP in samples from pregnant women and adolescents have to be taken into account. The assay is well suited for differential diagnosis of hepatic diseases in decentralized testing.

Rapid detection of the CYP2D6*3, CYP2D6*4, and CYP2D6*6 alleles by tetra-primer PCR and of the CYP2D6*5 allele by multiplex long PCR.

BACKGROUND: Interindividual differences in CYP2D6 activity range from total absence of metabolism of certain drugs to ultrafast metabolism and can produce adverse effects or lack of therapeutic effect under standard therapy. Several mutations have been described in the CYP2D6 gene that abolish CYP2D6 activity. However, four mutations explain the majority of the poor metabolizers. We describe four single-tube assays to detect these mutations. METHODS: Three tetra-primer PCR assays were developed to detect the mutations in the CYP2D6*3, *4, and *6 alleles. In these single-tube assays, the CYP2D6 locus is amplified directly, followed by the allele-specific amplification on this new template. In addition, a multiplex long PCR was developed to genotype the CYP2D6*5 allele. Two long PCR amplifications for detection of the deletion of CYP2D6 (*5) and for detection of the CYP2D6 gene region were combined in one tube. RESULTS: Analysis of 114 alleles showed no CYP2D6*3 allele, and allele frequencies of 28.1% for CYP2D6*4, 2.6% for CYP2D6*5, and 0. 9% for CYP2D6*6. Re-analysis of the DNA samples by restriction fragment length polymorphism and sequencing analysis confirmed these results. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7-11 times) always showed identical results. CONCLUSIONS: Our set of single-tube assays allows rapid and reproducible genotyping of the majority of CYP2D6 poor metabolizers.

Estimating imprecision profiles in biochemical analysis.

We describe a computer program IMPROFIL which determines an imprecision profile of an analytical method from replicated measurements of samples. It calculates the variance function, the coefficient of variation, the power of definition, the critical limit, the limit of detection and the lower limit of quantification. The primary property, the variance function, is determined by two alternative methods: the conventional maximum approximate conditional likelihood method and the newly developed weighted absolute deviation method. For all quantities, confidence intervals are obtained using the bootstrap procedure. The program combines the use of robust numerical techniques, user-friendliness and integration into a spreadsheet program for data pre- and post-processing. The algorithms used are described in detail. Tests with synthetic data sets are used to validate the method and to establish its powers and limitations. Finally, its application to a practical analytical task (tumor marker CA 15-3 in human sera) is reported. For the method to yield a reliable estimate of the variance function and the derived properties, certain minimum requirements on the raw data must be met: They have to be spread throughout the concentration range of interest, there should not be less than three replicates per specimen, and there must be at least of the order of 25 (better at 50) specimens.

Specific determination of germ cell alkaline phosphatase for early diagnosis and monitoring of seminoma: performance and limitations of different analytical techniques.

Two-dimensional electrophoresis, ion-exchange chromatography and immunoassay were evaluated in order to improve the diagnostic specificity of the germ cell specific isoenzyme of alkaline phosphatase (GCAP) for the detection of seminoma. Assessment of GCAP is hampered by its structural heterogeneity and low serum concentration. The structural heterogeneity of GCAP from seminoma tissue could be clearly visualized by two-dimensional electrophoresis. We inferred that it depended on allelic amino acid substitutions, varying sialylation and differential cleavage of the membrane anchor. The allelic variability of GCAP affects the accuracy of immunological measurements. However, immunoassay was found to be the only technique sensitive enough to assess GCAP in serum. The elevated GCAP levels in 15% of healthy blood donors were shown to be correlated with smoking. Further studies clarifying how to interpret the values measured in smokers are prerequisite for the introduction of GCAP as a serum marker for seminoma. In the future, GCAP might be utilized for the detection of carcinoma in situ (CIS) cells in ejaculate. Assessment of the enhanced expression of cellular GCAP by CIS cells exfoliated into ejaculate could be a means for noninvasive, early diagnosis that presumably will not be hampered by the patient's smoking habits.

Pharmacokinetic studies of mitoxantrone and one of its metabolites in serum and urine in patients with advanced breast cancer.

OBJECTIVE: Mitoxantrone (MTO) was administered to patients with advanced breast cancer either as free MTO (f-MTO) or liposomal MTO (1-MTO). The intra- and interindividual variations in serum pharmacokinetics of MTO were analysed. In addition, the excretion of MTO and its metabolite mitoxantrone dicarboxylic acid (MTOD) in urine was determined. METHODS: The concentration of MTO was measured by high-performance liquid chromatography in serum over a period of 24 h and the amount of MTO and the metabolite MTOD excreted in urine over 18 h was determined. Pharmacokinetic parameters of f-MTO and 1-MTO were calculated. RESULTS: 1-MTO had a significantly longer half-life of distribution in the deep (third) compartment and thus a larger area under the curve (AUC) than f-MTO. No difference was found with respect to distribution in the peripheral (second) compartment. The kinetics of MTO in serum did not significantly differ between patients. In four patients repeated pharmacokinetic analyses gave superimposable results. Thus, there was no enzyme induction during therapy. By contrast, two patients with oedema had a much longer mean residence time (MRT) and AUC for MTO in serum. Despite the altered pharmacokinetics of f-MTD and 1-MTO, no toxic adverse effects occurred in these two patients. CONCLUSIONS: f-MTO and 1-MTO exhibited different distribution patterns in the deep compartment with a significantly increased half-life for 1-MTO. There is no need to monitor MTO for treatment of breast cancer patients with f-MTO. In patients with oedema, the MRT of MTO is prolonged. The clinical relevance of this observation is as yet unclear.

[External quality control in the medical laboratory: evaluation of 12 ring trials 1994-1996]. / Externe Qualitätskontrolle im medizinischen Labor: Auswertung von 12 Ringversuchen der Jahre 1994-1996.

The regulations for reimbursement of laboratory tests provide that such tests will be paid for by social insurance institutions only if the laboratories participate in internal and external quality control schemes. Twelve surveys of the external quality assessment scheme of MQ Zürich (Association for Medical Quality Control) were evaluated. We analyzed the imprecision and inaccuracy of the participant results depending on the analytical system or methods used. Furthermore, the number of participants who met the quality criteria published by FKGRAL (Expert Committee for Overall Revision of Analysis List) was determined. The deviations from the internationally recommended or reference methods respectively were within +/-33% for the metabolites and enzymes if native plasma was used as control sample. If lyophilized samples were used, 5 deviations observed were > +/-33% (maximum +99%). For hematologic parameters the deviations were in the range of +/-10%. The CV's were 5.7-17.6% for wet chemistry methods used by the participants and 4.5-15.1% for the other methods. (Cobas Ready, Ektachem, Reflotron, Vision). For hematologic parameters we found CV's between 4.5 and 14.0%. 69-83% of the participants using wet chemistry methods met the FKGRAL criteria, while 86-98% of participants using one of the other system obtained adequate results. The corresponding figure for the hematologic parameters was 83-93%. The nature of the control samples (native samples or lyophilizate) did not influence the number of participants who successfully passed the survey. The study showed that the surveys are an adequate tool for determining participants with inadequate analytical performance, and in many cases the survey results make it possible to propose the necessary educative measures.

Pharmacokinetics of N4-octadecyl-1-beta-D-arabinofuranosylcytosine in plasma and whole blood after intravenous and oral administration to mice.

N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) is a new cytotoxic derivative of cytosine arabinoside with improved cytotoxic activity and stability against deamination. Its pharmacokinetics were studied in mice. The drug was administered intravenously and orally to ICR mice to assess its pharmacokinetic parameters in plasma and whole blood. The lipophilic drug was administered in small unilamellar liposomes 100-400 nm in diameter. The concentrations of NOAC in plasma and erythrocytes were determined by high-performance liquid chromatography (HPLC). When given orally a rather low amount of the delivered NOAC was absorbed as the unchanged drug, resulting in a bioavailability of 1.1% from the plasma and 12.9% from whole blood. As shown elsewhere, the amount of drug absorbed is sufficient to provide excellent cytotoxic activity in the L1210 leukemia and in human xenograft models after oral administration. The mean residence time of NOAC after intravenous administration was 3.5 h in plasma and 6 h in whole blood giving NOAC a terminal half-life in blood substantially longer than that of cytosine arabinoside. After oral administration the mean residence time was 18 h in plasma and whole blood. In summary, NOAC has a prolonged half-life after intravenous administration compared with cytosine arabinoside. The distribution of NOAC in blood is highly dependent on its mode of administration.

Simultaneous detection of all four alkaline phosphatase isoenzymes in human germ cell tumors using reverse transcription-PCR.

We have developed a reverse transcription-PCR method that clearly distinguishes between the RNA transcripts of all four alkaline phosphatase (AP) genes. If compared to the methods used up to the present, the main advantages of the reverse transcription-PCR method presented are its specificity and high sensitivity. The germ cell AP and the placental AP, which are the two most closely related AP isoenzymes (98% homology), can clearly be distinguished without any interference by other AP isoenzymes. An enhanced expression of AP isoenzymes has been reported for various tumors. The examination of the pattern of AP isoenzyme expression in a specific tumor and the corresponding tissue of origin enables discrimination between eutopically and ectopically expressed isoenzymes and thus represents an important tool in the elucidation of AP isoenzymes as potential tumor markers. The pattern of AP expression in 15 germ cell tumors, 2 germinal epithelia adjacent to seminoma, 2 cell lines of germ cell tumor origin (Tera-1 and BeWo), and 5 normal testes was studied. In comparison to normal testes, in all seminomatous germ cell tumors eutopic expression of germ cell AP and ectopic expression of tissue-nonspecific AP were demonstrated. In both samples of pure embryonal carcinoma and in the embryonal carcinoma cell line, the transcription of all four mRNAs was shown. These results indicate that the expression of the isoenzymes depends on the degree of differentiation of a tumor and that a simultaneous up-regulation of all AP isoenzymes in all types of germ cell tumors does not exist.

Lactate dehydrogenase-elevating virus (LDV): lifelong coexistence of virus and LDV-specific immunity.

Viruses have developed various strategies to coexist with vertebrate hosts. Lactate dehydrogenase-elevating virus (LDV) is a highly cytopathic virus exhibiting an extraordinary rate of replication; LDV nevertheless establishes a persistent infection without harming the host. The cytotoxic and helper T cell responses to LDV were monitored in mice with different genetic backgrounds. LDV-specific cytotoxic and helper T cells were found in all strains tested. These responses persisted for at least up to 250 days despite high levels of LDV in the blood. Thus, the cytopathic LDV induces and maintains an inefficient immune response that is not exhausted. LDV infection in mice reveals a special type of host-virus equilibrium where LDV quickly establishes persistence despite continuously induced LDV-specific helper and cytotoxic T cell responses, which apparently are too slow to control the highly cytopathic and extremely fast replicating virus.

European multicentre evaluation of the analytical performance of the Abbott AxSYM Abused Drugs Assays.

In accordance with the guidelines of the European Committee for Clinical Laboratory Standards (ECCLS), the performance of the Abbott AxSYM Abused Drugs assays were evaluated and compared with the results provided by the following systems: Syva Emit d.a.u./Roche Cobas Mira S Plus, Abbott TDx and ADx, Syva Emit d.a.u./Syva ETS Plus, Syva Emit II/Hitachi 717 and Roche Abuscreen OnLine/Roche Cobas Mira S Plus. The test analytes, cannabinoids, cocaine metabolites, opiates, benzodiazepines and barbiturates, were each investigated in three laboratories on different systems. The imprecision of all systems in the series and from day to day was good, with CVs less than 5% or 10%, respectively. The AxSYM calibration curves were stable for 3-4 months and none of the systems displayed any shift in the results of the analyses within one day or any faults caused by sample contamination. Within the framework of this study, a total of 1860 urine samples were investigated; 741 results were positive. All results which remained discrepant between AxSYM and the comparison systems after repeated analysis (n = 17) were subjected to further investigation using a reference method, with the exception of one barbiturate and two benzodiazepine samples. An additional test criterion was the practicability of the systems investigated and the versatility of the software. During this evaluation, the results provided by the Abbott AxSYM were excellent and were fully in line with the manufacturer's claims. The reliability of the FPIA technology that has been the subject of frequent investigation was also convincing during this evaluation. The possibility of semi-quantitative determination, the stability of the calibration curves, the ability to process an emergency sample without delay and its high suitability to routine operations are the convincing benefits offered by this system.

Comparative pharmacokinetic and cytotoxic analysis of three different formulations of mitoxantrone in mice.

Two liposomal formulations of mitoxantrone (MTO) were compared with the aqueous solution (free MTO) in terms of their pharmacokinetic behaviour in ICR mice and cytotoxic activity in a nude mouse xenograft model. The three different formulations of MTO [free MTO, phosphatidic acid (PA)-MTO liposomes, pH-MTO liposomes] were administered intravenously (three mice per formulation and time point) at a dose of 4.7 micromol kg(-1) for free MTO, 6.1 micromol kg(-1) for PA-MTO and 4.5 micromol kg(-1) for pH-MTO. The concentrations of MTO were determined using high-performance liquid chromatography (HPLC) in blood, liver, heart, spleen and kidneys of the mice. Additionally, the toxicity and anti-tumour activity of MTO was evaluated in a xenograft model using a human LXFL 529/6 large-cell lung carcinoma. The dose administered was 90% of the maximum tolerated dose (MTD) of the corresponding formulation (8.1 micromol kg(-1) for free MTO, 12.1 micromol kg(-1) for PA-MTO and pH-MTO). The pharmacokinetic behaviour of PA-MTO in blood was faster than that of free MTO, but the cytotoxic effect was improved. In contrast, pH-MTO showed a tenfold increased area under the curve (AUC) in blood compared with free MTO, without improvement of the cytotoxic effect. This discrepancy between the pharmacokinetic and cytotoxic results could be explained by the fact that MTO in pH-MTO liposomes remains mainly in the vascular space, whereas MTO in PA-MTO liposomes is rapidly distributed into deep compartments, even more so than free MTO.

Estimation of the lower limits of manual and automated platelet counting.

Most evaluators of automated or manual methods for platelet counting focus on characteristics such as imprecision, linearity, and carry over. The limits of the analytical procedure are usually not assessed. The limits of the different techniques are neither discussed in the literature nor do manufacturers of analytical systems supply these data. A new procedure is presented to assess the performance of the manual as well as the automated platelet count. This procedure allows, with defined statistical confidence (eg, 95%), the determination of (1) the limit of platelet detection (LD) at which signals of platelets can be discriminated from the system noise; (2) the lower limit of quantification (LLQ), at which a certain imprecision is not surpassed; and (3) the power of definition (PD) that defines the number of values that can be discriminated in a certain interval. For each value, the PD allows calculation of the two adjacent (lower and higher) values that are significantly (P > or = 0.95) different. For the manual count, LD was found to be 1.6 x 10(9) plt/L and the LLQ 6.9 x 10(9) plt/L. For the automated count with the Technicon H1, LD was 4.3 x 10(9) plt/L and LLQ 13.8 x 10(9) plt/L (CVmax = 15%). The PD in the range 20 to 100 x 10(9) plt/L is 8 for the automated and 7 for the manual count.

Determination of mitoxantrone in mouse whole blood and different tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method was developed for the specific determination of mitoxantrone (MTO) in whole blood and different tissues of mice (liver, heart, spleen, kidneys). MTO was extracted into dichloromethane with ametantrone (AMT) as internal standard. The different tissues were homogenised in citrate buffer (pH 3.0) containing 20% ascorbic acid. Separation of MTO and AMT was carried out using a Nucleosil C18 column. The mobile phase consisted of acetonitrile (33%) and 0.16 M ammonium formate buffer, pH 2.7. UV detection was used at 658 nm. Baseline separation of AMT and MTO was achieved in all matrices. The calibration curves were linear in all matrices (r > 0.999) in the concentration range of 2-200 micrograms/l for whole blood and 2-700 micrograms/l for tissue homogenates, respectively. The within-day and between-day precision studies showed good reproducibility with coefficients of variation below 4.5% for whole blood and below 10% for tissue homogenates, respectively. The extraction efficiencies of MTO are 60% in whole blood and 38% in tissue homogenates. The method described is suitable for pharmacokinetic studies on the distribution of MTO in different tissues of mice.

Sensitive high-performance liquid chromatographic method for the determination of N4-hexadecyl- and N4-octadecyl-1-beta-D-arabinofuranosylcytosine in plasma and erythrocytes.

N4-Hexadecyl- and N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NHAC, NOAC) are two new cytostatic derivatives of cytosine arabinoside (ara-C) with improved cytostatic activity and stability against deamination. A high-performance liquid chromatography (HPLC) method was developed for the specific determination of NHAC and NOAC in plasma and erythrocytes, after solid-phase extraction using UV detection at 275 mm. Because of the strong binding of the drugs to proteins and membranes, the samples have to be pretreated with urea (plasma) or butanol and ultrasonication (erythrocytes). The calibration curves are linear for both drugs (r > 0.999) in the concentration ranges 20-2100 micrograms/l for plasma and 40-4200 micrograms/l for erythrocytes, respectively. The within-day and between-day precision studies showed a good reproducibility, with coefficients of variation below 8.5%. The recoveries of the lipophilic ara-C derivatives are greater than 66%. The method described can be applied to pharmacokinetic studies with NHAC and NOAC.