Molecular mechanisms of ethanol biotransformation: enzymes of oxidative and nonoxidative metabolic pathways in human.

Ethanol, as a small-molecule organic compound exhibiting both hydrophilic and lipophilic properties, quickly pass through the biological barriers. Over 95% of absorbed ethanol undergoes biotransformation, the remaining amount is excreted unchanged, mainly with urine and exhaled air.The main route of ethyl alcohol metabolism is its oxidation to acetaldehyde, which is converted into acetic acid with the participation of cytosolic NAD+ - dependent alcohol (ADH) and aldehyde (ALDH) dehydrogenases. Oxidative biotransformation pathways of ethanol also include reactions catalyzed by the microsomal ethanol oxidizing system (MEOS), peroxisomal catalase and aldehyde (AOX) and xanthine (XOR) oxidases. The resulting acetic acid can be activated to acetyl-CoA by the acetyl-CoA synthetase (ACS).It is also possible, to a much smaller extent, non-oxidative routes of ethanol biotransformation including its esterification with fatty acids by ethyl fatty acid synthase (FAEES), re-esterification of phospholipids, especially phosphatidylcholines, with phospholipase D (PLD), coupling with sulfuric acid by alcohol sulfotransferase (SULT) and with glucuronic acid using UDP-glucuronyl transferase (UGT, syn. UDPGT).The intestinal microbiome plays a significant role in the ethanol biotransformation and in the initiation and progression of liver diseases stimulated by ethanol and its metabolite - acetaldehyde, or by lipopolysaccharide and ROS.

Serum cortisol concentration in patients with major depression after treatment with fluoxetine.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and elevated cortisol levels is characteristic of the pathophysiology of major depressive disorder (MDD). The aim of this study was to determine whether increased plasma cortisol levels appear in patients with major depression and if effective antidepressant treatment by fluoxetine leads to regulation of cortisol level. This aim was realized by describing and validation of methods of determining fluoxetine and cortisol in serum and searching for correlation between their concentrations in patients with endogenous depression, the therapeutic effect as assessed in Hamilton Depression Rating Scale (HDRS), age and sex of patients. Plasma cortisol and fluoxetine levels were measured using high performance liquid chromatography (HPLC) methods with applying Shimadzu chromatograph with UV detection. Plasma cortisol and fluoxetine levels were measured at time zero (before therapy) and after 6h, 24h, 2, 4, 6 and 8 weeks of fluoxetine administration in patients with major depression qualified for therapeutic drug monitoring (TDM). The study included 21 patients (14 women, 7 men; mean age 29-75 years) and 24 healthy comparison subjects. The patients had a mean score on the 21-item HDRS. As the effect of fluoxetine administration the decrease of the level of cortisol was observed in patients who responded to the therapy (the reduction of points in HDRS scale in at least 50%). The validation parameters of HPLC method of fluoxetine and cortisol determination indicate the possibility of applying them for determination of both: the level of concentration of the drug in therapeutic drug monitoring and the level of cortisol in serum of patients with endogenous depression.

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. IV. Application of long polymerase chain reaction for identification of CYP2D6 gene duplication.

In the human organism 58 cytochrome P450 (CYP) isoenzymes belonging to 18 families have been described. Isoenzyme CYP2D6 is an important human xenobiotic-metabolizing enzyme. CYP2D6 biotransforms a significant number of drugs, widely used in clinical practice, such as antidepressants, neuroleptics, antiarrhythmics, analgesics, antiemetics and anticancer agents. The occurrence of polymorphic variants of the enzyme results in different metabolic capacity ranging from poor to ultrarapid. Ultrarapid metabolizer phenotype explains lack of response and decreased levels of drugs which are metabolized by CYP2D6. Therefore, the identification of ultrarapid metabolizers as potential non-compliance cases requiring dose adjustment, has serious clinical importance. In this study we evaluate a long-PCR procedure for detecting CYP2D6 gene duplication.

Urinary proteins, N-acetyl-ß-D-glucosaminidase activity and estimated glomerular filtration rate in hypertensive patients with normoalbuminuria and microalbuminuria.

BACKGROUND: The aim of the study was to assess novel candidate markers measured in the urine of normoalbuminuric and microalbuminuric patients (the urinary albumin-to-creatinine ratio < 30 mg/mmol) with essential hypertension to be used for early detection and assessment of progressive deterioration in renal function. METHODS: Albumin, α-1-antitrypsin, orosomucoid, transferrin, retinol-binding protein and α-1-microglobulin concentrations and the NAG (N-acetyl-ß-D-glucosaminidase) activity in the urine were evaluated in 102 hypertensive subjects with urinary albumin-to-creatinine ratio (uACR) < 30 mg/mmol. The estimated glomerular filtration rate (e-GFR) was calculated using the Modification of Diet in Renal Disease Study equation. RESULTS: The decreasing e-GFR values in normo- and microalbuminuric patients with essential hypertension were accompanied by significant increases (P < 0.05) in the NAG activity and uACR value in the urine. The e-GFR significantly (P < 0.05) correlated with the NAG activity in the urine, but no association was observed with the urinary concentrations of any of the individual proteins (P > 0.05). CONCLUSIONS: In normoalbuminuric and microalbuminuric patients with essential hypertension renal impairment measured by e-GFR is related to the increased urinary NAG activity and uACR rather than elevated concentrations of individual proteins. Urinary NAG activity and uACR value seem independently promising candidate markers for use in assessing progression of early renal impairment in patients with hypertension.

Simultaneous determination of eight estrogens and their metabolites in serum using liquid chromatography with electrochemical detection.

The liquid chromatography (LC) with electrochemical detection allows to determine, evaluate and validate the level of estrogens and their metabolites in serum. The method is fast and sensitive, and the hormones can be determined simultaneously, from one serum sample. The proposed method was successfully applied to the determination of following estrogens: estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)) and following catecholestrogens: 2-hydroxyestradiol (2-OHE(2)), 4-hydroxyestradiol (4-OHE(2)), 4-hydroxyestrone (4-OHE(1)), 2-methoxyestradiol (2-MOE(2)) and 2-methoxyestrone (2-MOE(1)). The method of LC with electrochemical detection (LC/EC) was applied for the determination of catecholestrogens in serum sample taken from pregnant women. Estrogens and catecholestrogens were extracted from 1 mL of serum by applying diethyl ether under the specified conditions. The parameters of the procedure included using a specific mobile phase, applying the column Symmetry C18 (5 microm, 3.0 mm x 150 mm), equipping the applied electrochemical detector with the working glassy-carbon electrode, as well as applying the reference electrode Ag/AgCl. The calibration studies on this study were performed, and a good analytical performance for E(1), E(2), E(3), 2-OHE(2), 4-OHE(2), 4-OHE(1), 2-MOE(2) and 2-MOE(1) was attained, along with low limits of detection (LOD of 0.18-0.30 ng/mL), satisfactory limit of quantification (LOQ of 0.23-0.92 ng/mL) and excellent linear dynamic range (0.6-8.0 ng/mL). In conclusion, the presented methodology is the sensitive method of the simultaneous measurement of eight estrogens and their metabolites from one sample of blood and it might be clinically applied for testing serum of pregnant women, as well as for further studies on estrogens and their metabolites.

Recombinant adeno-associated viruses (rAAV2) facilitate the intraperitoneal gene delivery to cancer cells.

Peritoneal dissemination of cancer cells is characteristic of advanced stages of ovarian, breast and lung cancers, and is associated with poor patient survival. The presence of cancer cells in effusions complicates treatment protocols, while cell eradication is seriously limited. One of the novel options available is cancer gene therapy with recombinant adeno-associated viruses. This combination represents the most promising gene delivery vehicles to neoplasmatic cells within serosal cavities due to their unique properties that include the ability to infect proliferating cells of broad host range, as well as the potential of long-term expression. Recombinant infectious adeno-associated virus serotype 2 particles (rAAV2) were produced in a helper-free system using an AAV-293 packaging cell line, and quantitatively analyzed by real-time PCR. Balb/c mice intraperitoneally pre-injected with L1 cancer cells were treated with different doses of rAAV2. Subsequently, the mice were sacrificed and intraperitoneal effusions were analyzed for rAAV presence and rAAV/ß-galactosidase (LacZ) vector efficiency in order to infect cancer cells within the peritoneal cavity. We reported an efficient infection of L1 cancer cells disseminated into the peritoneal cavity by rAAV2. The expression of reporter genes (GFP and LacZ) attributable to the rAAV cell uptake was closely dependent on an applied multiplicity of infection ratio (MOI). The highest infection efficiency was observed at a MOI of 50 and 200. Our study confirmed the ability of adeno-associated viruses to facilitate gene transferability to cancer cells disseminated in the serosal cavity, as well as the potential usefulness of these viruses as a new approach in cancer gene therapy.

Serum cortisol concentration in patients with major depression after treatment with clomipramine.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and elevated cortisol (CORT) levels are characteristics of the pathophysiology of major depressive disorder. The aim of this study was to determine whether increased plasma CORT levels appear in patients with major depression and if effective antidepressant treatment by clomipramine (CLO) leads to regulation of CORT level. Plasma CORT levels were measured using high performance liquid chromatography (HPLC) methods in patients with major depression at time zero (before therapy) and after 3 h, 24 h, 4, 6 and 8 weeks of CLO administration. The study included 17 patients (12 women, 5 men; mean age 54.5 years, SD =12.3) and 21 healthy comparison subjects. The patients had a mean score on the 21-item Hamilton Depression Rating Scale (HDRS) of 26.8 (range 22-35). Eight of the patients with major depression recruited for the study showed a 46% increase in CORT concentration compared to the established standard. In 13 patients treated with CLO, serum CLO levels reached a therapeutic range. In recovered depressed patients, antidepressant treatment significantly reduced HDRS scores from the 6th week of treatment. A drop in plasma CORT levels in recovered depressed subjects occurred 0 to 6 weeks after CLO treatment (n = 5, p < 0.046). However, neither subject group exhibited any definitive markers of CORT secretion. In the population studied, patients had distinct profiles of HPA axis dysregulation. Finding a linear correlation between lower CORT secretion and therapeutic plasma CLO levels is the first aim of monitored therapy and may be important for understanding the pathophysiology of major depressive disorder.

Recombinant adeno-associated virus derived vectors (rAAV2) efficiently transduce ovarian and hepatocellular carcinoma cells--implications for cancer gene therapy.

Recombinant adeno-associated virus vectors (rAAV) represent a most promising gene delivery vehicles for gene therapy applications because their unique properties, such as capability to infect both proliferating and non proliferating cells of broad host range, and possibilities of long-term expression and site-specific integration. rAAV are also described as vectors neither toxic nor pathogenic to the cells. rAAV vectors are also thought to be attractive for cancer gene therapy. Here, we used rAAV2 vectors encoding reporter genes, rAAV/GFP and rAAV/LacZ to transduce cancer cells. The rAAV preparations were produced by a transient triple AAV plasmid transfection of AAV-293 packaging cells and isolated/purified by iodixanol-gradient method. We report a different rAAV transduction efficiency of the two cancer cell lines cells--ovarian carcinoma (OVP 10) and hepatocellular carcinoma (C3A) cells. The expression of the reporter genes due to rAAV uptake was about two fold higher for ovarian cells than for hepatocellular cells. Our studies have also revealed the long-term expression of GFP gene in hepatocellular (C3A) rAAV/GFP transduced cells. These findings indicate that adeno-associated virus derived vectors could be very useful for cancer gene therapy applications, however, further investigations of the mechanisms of rAAV gene delivery are still needed.

Cytochrome P450 polymorphism--molecular, metabolic and pharmacogenetic aspects. I. Mechanisms of activity of cytochrome P450 monooxygenases.

Cytochrome P450, initially perceived as a type of cell pigment, was soon identified as a hemoprotein with an enzymatic activity characteristic for monooxygenases with an affinity for differentiated endo- or exogenous substrates, including drugs. So far in the human organism 58 CYP isoenzymes belonging to 18 families have been described. Most from the CYP monooxygenases superfamily turned out to be integral elements of hepatocytic reticular monooxygenase complexes which also contain NADPH-dependent cytochrome P450 reductase (CPR). Later investigations indicated the possibility of the participation in electron transport for reticular CYP isoenzymes, alternative NADH-dependent reticular system composed of cytochrome b5 reductase (CBR) and cytochrome b5. The demonstration of the activity of some CYP superfamily isoenzymes not only in hepatocytes but also in many other cells of the human organism, numerous plant and animal tissues and even in cells of fungi, protists and prokaryotes has contributed to the significantly increased understanding of the role of CYP in biological systems. In addition, some CYP isoenzymes were found to be characteristic for the inner mitochondrial membrane monooxygenase complexes which contain NADPH-dependent adrenodoxin reductase (AR) and adrenodoxin (Ad), which is identical with ferredoxin-1 (Fd-1) and hepatoredoxin (Hd).

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. II. Participation of CYP isoenzymes in the metabolism of endogenous substances and drugs.

In the human organism 58 cytochrome P450 (CYP) isoenzymes belonging to 18 families have been described. These hemoproteins, with enzymatic activity characteristic for monooxygenases, show a broad affinity for chemically differentiated endo- or exogenous compounds, including drugs. CYP isoenzymes participate in metabolic pathways important for proper physiological functioning of the human organism, i.e.: cholesterol, bile acid and oxysterol biosynthesis; metabolism of fatty acids, prostaglandins, prostacyclins, leukotrienes, steroid hormones, ketone bodies, vitamines A and D. CYP isoenzymes participate in the metabolism of over 80% of drugs and other xenobiotic substances which can be present in the human organism. Differences in molecular structure and kinetics of conformational changes of particular isoenzymes of CYP superfamily monooxygenases on the one hand determine their affinity direction for chemically differentiated groups of compounds susceptible to oxidation, on the other hand determine the mechanism and position of the oxidative change of their molecules. Drugs and their metabolites and other endogenous and xenobiotic compounds may be acting not only as substrates, but also as competitive and non- competitive inhibitors, suicide inhibitors and inducers of CYP isoenzymes as well as repressors of CYP genes. These relationships are the metabolic basis of numerous multidirectional interactions between drugs, drug metabolites, food components, stimulants, environmental toxins and metabolites of these xenobiotics.

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. III. Influence of CYP genetic polymorphism on population differentiation of drug metabolism phenotype.

In the human genome 684 alleles of CYP genes, and additionally 30 complete CYP pseudogenes, have been identified. So far 388 isoforms of 58 human CYP isoenzymes have been described at the phenotypic level. The molecular forms of many CYP isoenzymes responsible for drug biotransformation show a differentiated degree of specific catalytic activity - from increased, through normal and decreased to various extent, to trace or even absent. Depending on the homo- or heterozygous genotype, a broad palette of phenotypic forms may be present, differentiated in respect to biotransformation dynamics of specific drugs. The progress of molecular biology with particular consideration of genotyping and DNA microarray technologies has created a basis for the dynamic progress of pharmacogenetics, allowing fast and sensitive determination of the individual pharmacogenetic profile, encompassing a large set of CYP alleles extended by allelic variants of genes encoding other enzymes participating in drug metabolism. The possibility to evaluate the pharmacogenetic profile of patients together with the increasing knowledge about the mechanisms of inhibition, repression and also induction of enzymes participating in biotransformation of xenobiotics and endogenous compounds create increasing possibilities of elaborating optimal individualized pharmacotherapeutic strategies.

Cortisol level in men with major depressive disorder treated with fluoxetine or imipramine.

The aim of this research was to find out whether increased plasma cortisol levels appear in unipolar or bipolar patients with major depressive disorder (MDD) and whether the effective antidepressant treatment by imipramine and fluoxetine leads to regulation of the cortisol level. Cortisol levels were studied in two groups of patients with major depressive disorder: unipolar and bipolar patients treated with fluoxetine (doses: 20-60 mg/day). This group included 5 patients (age 29-46 yr); unipolar and bipolar subjects treated with imipramine (50-150 mg/day), this group included 5 patients (aged 24-70 yr). Cortisol and fluoxetine or imipramine plasma levels were assessed using HPLC methods: before treatment, after 3, 6 and 24 h of drug administration as well as in the 2nd, 4th, 6th, and 8th week of antidepressant treatment. HPLC methods were previously validated. The research conducted and the clinical data may be useful for proving the essential role of enhanced HPA axis activity for the pathogenesis and depressive disorder proceedings.

Development, validation and application of the HPLC method for determination of mianserin in human serum.

A high performance liquid chromatography method for the determination of mianserin in human serum was developed and validated. Doxepin was used as an internal standard. Mianserin was extracted from human serum using a liquid-liquid extraction with hexane:isoamyl alcohol (99:1, v/v). The sample was then dissolved in 0.05 M phosphoric acid (pH=3.0), and after separation on a Hichrom RPB (250 x 4.6 mm, 5 mm) column, the analytes were measured by ultraviolet detection at 214 nm. The recovery ranged from 86.1 to 94.5% for mianserin. The method was specific and linear over the concentration range of 2.0-128.0 ng/mL. The limit of quantification (LOQ) was established at 2.0 ng/mL (CV=13.8%). The accuracy range was from 92.5 to 107.5%. The method was used to measure mianserin in human serum samples obtained from healthy volunteers who had received a single oral dose of 30 mg mianserin. Pharmacokinetic parameters obtained for the mianserin were in agreement with the existing data.

Alpha-1-antitrypsin and IgA in serial meconium and faeces of healthy breast-fed newborns.

BACKGROUND: Meconium is a series of layers formed in the foetal intestine from the 12th week of gestation. High content of meconial alpha-1-antitrypsin (AAT), decreasing within the first several days of extrauterine life appears to reflect the meconium clearance of the gut. At birth, IgA is not present in the meconium and breast-fed infants receive this antibody postnatally with human milk. The aim of the study was to determine changes in AAT concentrations, functional activity of that inhibitor expressed as trypsin inhibitory capacity (TIC) and IgA concentration in serial meconium and faeces, as endogenous biochemical markers discriminating between faeces portions formed in intrauterine and extrauterine life periods of healthy breast-fed newborns. METHODS: A group of 24 healthy breast-fed newborns delivered by spontaneous labour were studied prospectively during the first 4 days of postnatal life. AAT and IgA concentrations in the newborn's meconial and faecal samples and IgA concentration in mother's milk samples taken on the third day after delivery, were determined by radial immunodiffusion. TIC was assessed using BAPNA (N-benzoyl-DL-arginine-p-nitroanilide). RESULTS: The medians (range) of AAT concentrations in milligrams per gram of dry meconium or faeces were: 68.8(29.2-138.4) (day 1), 56.9 (30.8-112.8) (day 2), 26.2 (6.8-80.7) (day 3), and 6.6 (1.4-27.1) (day 4). The medians (range) of TIC in milligrams of trypsin/g dry mass of meconium or faeces were: 0.76 (0.33-1.79) (day 1), 0.44 (0.17-1.08) (day 2), 0.16 (0.03-0.56) (day 3), and 0.03 (0-0.11) (day 4). The median (range) of IgA concentration in mothers' milk was 715 mg/dl (420-890). IgA was absent in meconium portions from the first day of life while on the successive days the medians (range) of IgA concentration in mg/g dry mass of meconium and faeces were as follows: 0 (0-2.90) (day 2), 2.50 (1.10-9.60) (day 3), 7.05 (4.10-30.60) (day 4). On day 4 of extrauterine life a negative correlation was found between AAT and IgA concentrations in faeces of the newborns (r = -0.46) and a positive correlation was seen between IgA concentrations in faeces and milk (r = 0.93). CONCLUSIONS: Analyses of the systematic decrease in AAT and increase of IgA concentration in serial portions of meconium and faeces over the first days of extrauterine life of breast-fed newborns can date newborn's faeces portions formed during intrauterine and extrauterine maturation. AAT deposited in foetal intestine is an active antiprotease.

Trypsin and antitrypsin activities and protein concentration in serial meconium and feces of healthy newborns.

BACKGROUND: Meconium has the potential for being the matrix on which markers of fetal exposure to physiologic and non-physiologic agents during intrauterine life can be analyzed. The aim of this study was to compare trypsin and antitrypsin activities and protein concentration during intra- and extrauterine human development based on an assessment of these parameters in serial meconium and first feces of healthy, term newborns during the first four days of life. METHODS: One hundred and eighteen mature, term newborns were studied. Single portions of meconium or feces were taken prospectively from day 1 to day 4. In ten newborns each meconium and feces passed by the infant from birth up to the fourth day after delivery was collected individually. Trypsin activity was measured using L-TAPA (N-alpha-tosyl-l-arginine-p-nitroanilide) as substrate, trypsin inhibitory capacity (TIC) with N-alpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate, and protein concentration by the method of Lowry et al. RESULTS: Meconium and feces of healthy newborns demonstrated a decrease in TIC during the first two days of life and an increase in trypsin activity over the first three successive days of life. No correlation was observed between the variability of both parameters. During the first three days of a newborn's life, significant correlation was observed between TIC and protein concentration in meconium and first feces. In the course of proteolytic activity changes in successive portions of meconium and feces, a transient peak occurs on the 2nd-4th day of life. CONCLUSIONS: The gradual decrease in protease inhibitor activity and low trypsin activity in successive portions of meconium from the first two days of extrauterine life may provide a retrograde chronological depiction of the course of the second and third trimesters of intrauterine life. Serial collection of feces over 2-4 days may provide a reflection of the dynamics of the postnatal increase in the newborn's pancreatic exocrine functions.

RP-HPLC determination of paraoxonase 3 activity in human blood serum.

The aim of the present work was to establish conditions for paraoxonase 3 (PON3) activity determination in human blood serum with simvastatin (SV) as a substrate. The activity of PON3 is considered as a good early predictor of susceptibility to premature atherosclerosis as well as of statin therapy effectiveness. The method used quantifies the SV and beta,delta-dihydroxyacid simvastatin (SVA) liberated from SV after incubation with blood serum, followed by deproteinization of the reaction mixture. Separation of SV and SVA was performed on an LC(18) column by isocratic elution with acetonitrile-K-phosphate buffer of pH 4.5 (v/v, 70:30) as a mobile phase at flow rate of 1.5 ml min(-1). Detection based on ultraviolet absorption at a wavelength of 239 nm was reliable for the simultaneous assay of SV and SVA. The applied method was sufficiently sensitive, precise and accurate for determination of low simvastatin lactone hydrolase (statinase) activity in blood serum of children (1.97-6.86 pmol min(-1) ml(-1)). The method is characterized by good linearity over the measurement range of 0.5-6 microg ml(-1) (1.194-14.3 nmol ml(-1)). Limits of detection (LOD) and quantitation (LOQ) for SV were 3.1 and 10.4 ng ml(-1), respectively. In case of SVA, LOD and LOQ were 4.7 and 14.44 ng ml(-) for a 20 microl sample, respectively. Precision and accuracy of PON3 statinase activity determination in human blood serum with SV as substrate were satisfactory and acceptable for bioanalytical methods.

A simpler and faster capillary electrophoresis method for determination of mianserin enantiomers in human serum.

A stereospecific sample stacking capillary zone electrophoresis method is described for determination of S(+) and R(-) enantiomers of mianserin (1,2,3,4,10,14b-hexahydro-2-methyldibenzo[c,f]pyrazino[1,2-a]azepine) in human serum. The enantiomers of mianserin were extracted from human serum in one step extraction procedure using the mixture n-heptane:ethyl acetate (80:20, v/v). After separation of layers and freezing at -28 degrees C the organic layer was decanted and evaporated under a stream of nitrogen. The sample was dissolved in the mixture: water:methanol:acetonitrile (2:1:1, v/v/v). Separation was conducted in an aqueous solution of phosphoric acid (0.075M) adjusted to pH = 3.0 with concentrated triethylamine, and 2 mmole/L of 2-hydroxypropyl-beta-cyclodextrin. The analytes were measured by ultraviolet detection at 214 nm after separation on a Fused-Silica eCAP capillary. Clozapine was used as an internal standard. Recovery of the enantiomers from serum ranged from 82.94 to 90.37%. Total time of analysis was 49 minutes, whereas the other methods needed up to 100 minutes.

Liquid chromatographic method for the determination of lidocaine and monoethylglycine xylidide in human serum containing various concentrations of bilirubin for the assessment of liver function.

A high-performance liquid chromatographic method is described for determination of lidocaine (2-(dietyloamino)-N-(2,6-dimetylofenylo) acetamid) and its metabolite, monoethylglycine xylidide (MEGX), in human serum containing various concentration of bilirubin. Lidocaine and its metabolite were extracted from human serum using dichloromethane. After separation of the layers and freezing at -32 degrees C, the organic layer was decanted and evaporated under a stream of nitrogen. The sample was dissolved in the mobile phase (12% acetonitrile in 15mM potassium dihydrogen orthophosphate, pH 3.0), and after separation on a Supelcosil LC-8-DB column, the analytes were measured by ultraviolet detection at 205nm. Trimethoprim (TMP) was used as the internal standard. The recovery of the examined analytes ranged from 95.7 to 97.9% for lidocaine and from 98.0 to 99.9% for MEGX. The lower limit of quantification (LLOQ) was established at 200microg/l for lidocaine and at 10microg/l for MEGX. The choice of suitable conditions for chromatographic separation of lidocaine and its metabolite MEGX allowed the elimination of the influence of endogenous bilirubin on the result of analysis.

New HPLC method for separation of blood plasma phospholipids.

The aim of the present work was to develop a new HPLC method for separation of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC) from small-volume samples of blood plasma. Human plasma glycerophospholipids were separated by liquid-liquid extraction method followed by solid phase extraction (SPE) on aminopropyl columns. Reversed-phase Sephasil C8 column (10 cm x 2.1 mm, I.D. 5 microm) and micropreparative chromatograph "SMART" were used for separation of PC, PE, LPC and PI from SPE phospholipids extract. Binary-step gradient of eluent A: acetonitrile-methanol (130:5, v/v) and B (0.01% trifluoroacetic acid) provided good, fast and reproducible resolution of investigated phospholipids classes in 12 min at 30 degrees C. Eluted phospholipids were detected at wavelengths lambda=235 and 254 nm. This method made it possible to determine quantitatively: 5 microg ml(-1) PC, 1 microg ml(-1) LPC, 4 microg ml(-1) PE and 3 microg ml(-1) PI in blood plasma samples.

Antigenic and functional levels of alpha-1-antitrypsin in serum during normal and diabetic pregnancy.

OBJECTIVES: Serum alpha-1-antitrypsin (AAT) concentration may not be representative of the functional capacity of this inhibitor. The aim of this study was to determine the antigenic and functional serum levels of AAT during normal and diabetic pregnancy. METHODS: Serum AAT concentration was measured on NOR-Partigen plates (Dade Behring). Trypsin inhibitory capacity (TIC) in the serum was determined with N-benzoyl-DL-arginine-p-nitroaniline (BAPNA, Sigma) as substrate. The examined material included pregnant women with type 1 diabetes mellitus (n=16) studied prospectively in successive stages of pregnancy, healthy pregnant women in the first trimester (n=12), second trimester (n=15), third trimester (n=15) and healthy non-pregnant women (n=14). RESULTS: Serum concentration of AAT in all consecutive phases of diabetic pregnancy is higher as compared to normal pregnancy (P<0.0001). Serum TIC is significantly lower in the first and third trimesters in diabetic pregnancy (P<0.05, 0.001, respectively). Specific activity of serum AAT (mg of trypsin inhibited by 1mg of AAT) does not change between subsequent trimesters both in normal and diabetic pregnancy and in diabetic pregnancy is two times lower as compared to normal pregnancy. CONCLUSION: In spite of the higher level of AAT in the serum in diabetic pregnancy, the ability of this inhibitor to inhibit trypsin is two times lower as compared to normal pregnancy.