Chromatofocusing of human high density lipoproteins and isolation of lipoproteins A and A-I.

Using chromatofocusing, a column chromatography method with an internally generated pH gradient and focusing effects, human plasma high density lipoproteins (HDL) were fractionated into six subclasses within an interval of less than 1 pH unit (pH 5.1-4.2). All fractions floated in the ultracentrifuge at density = 1.21 g X ml-1, retained a typical HDL electron micrographic morphology and as a single band, alpha-migration on agarose electrophoresis. Compositional analysis of the subclasses revealed an inverse relationship between cholesterol ester and cholesterol on a molar basis. Distinct differences in the distribution of the apolipoproteins between the fractions were found. Two of the subclasses contained only apolipoprotein A-I and were therefore considered to be two forms of the lipid-combined form of apolipoprotein A-I, i.e., lipoprotein A-I. One subclass contained only apolipoproteins A-I + A-II and was, therefore, lipoprotein A. One subclass contained apolipoproteins A-I + A-II + D, and the two remaining contained additionally apolipoproteins C and E. Lipoprotein A-I was also demonstrated after immunoabsorption of apolipoprotein A-II-containing lipoproteins from whole serum. It is suggested that this method, which allows the fractionation of HDL into subclasses with distinct differences in apolipoprotein composition, offers new avenues for the study of the structural and metabolic heterogeneity of HDL.

Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil.

1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.

Prenatal diagnosis of the Pallister-Killian mosaic aneuploidy syndrome by CVS.

Prenatal cytogenetic analysis at 11 weeks of gestation revealed an abnormal karyotype 47,XX,+mar in all metaphases obtained from a chorionic villi sample after 24 h culture. Karyotyping of amniotic fluid cells in the second trimester showed mosaicism 47,XX,+i(12p)/46,XX with 10% aneuploid cells. The pregnancy was terminated at 20 weeks of gestation on the patient's request. The aborted fetus showed typical manifestations of the Pallister-Killian mosaic aneuploidy syndrome. The identity of the supernumerary isochromosome 12p was proven by LDH isozyme electrophoresis using cultured fibroblasts and by nonradioactive in situ hybridization using a biotinylated set of chromosome 12-specific DNA probes.

Induction of cytokine release by the acyl glucuronide of mycophenolic acid: a link to side effects?

OBJECTIVES: We have identified an acyl glucuronide (M-2) of the immunosuppressant mycophenolic acid (MPA). Acyl glucuronides have toxic potential and may contribute to drug toxicity. Whether acyl glucuronides are able to induce release of proinflammatory cytokines is unknown. Gastrointestinal disturbances have been observed during MPA therapy and may involve an inflammatory reaction. This study investigated whether M-2 can induce IL-6 and TNF-alpha release as well as gene expression of these cytokines in leukocytes. DESIGN AND METHODS: M-2 was produced by incubation of MPA with human liver microsomes. Human mononuclear leukocytes were incubated in the presence of M-2. Concentrations of IL-6 and TNF-alpha were measured by ELISA. Expression of mRNA was determined by quantitative RT-PCR. RESULTS: Incubation of 3 x 10(6) cells with M-2 resulted in a time and dose dependent release of cytokines, whereas MPA or its phenolic glucuronide MPAG were without effect. Cytokine liberation depended on mRNA induction. Response to M-2 showed much inter individual variability (30-fold for IL-6, 3-fold for TNF-alpha). CONCLUSIONS: If M-2 promotes release of cytokines in vivo, these may mediate some of the toxic actions of MPA.

Suppression of leukocyte-enhanced cold ischemia/reperfusion injury of liver endothelium with the benzoquinone antioxidant idebenone.

OBJECTIVE: Despite the large body of evidence for a major role of neutrophils and oxidant stress, the exact pathogenesis of the early ischemia/reperfusion injury after cold preservation of the liver is not well understood. The potential benefit of an antioxidant on metabolic liver function during reperfusion has been demonstrated in several studies. MATERIALS AND METHODS: We describe a cold storage/reperfusion damage model with isolated perfused pig livers, where the effects of neutrophils and idebenone, a recently developed benzoquinone antioxidant were studied. The integrity of sinusoidal endothelial cells (SEC) was estimated by hyaluronic acid concentration in perfusate and the expression of endothelial constitutive nitric oxide synthase (ecNOS) after reperfusion and compared to lipid peroxidation and antioxidant content. RESULTS: Hyaluronic acid displayed the highest levels and ecNOS mRNA was most depressed in livers reperfused with neutrophils after 20 h cold storage; this was accompanied by an increase in lipid peroxidation (TBARS) and a breakdown of endogenous lipophilic antioxidants (alpha-tocopherol and coenzyme Q-10). These effects were attenuated, when neutrophils were excluded from reperfusion and almost completely abolished by the addition of 200 mumol/L idebenone. CONCLUSIONS: These data suggest that a leukocyte-mediated damage based on reactive oxygen species markedly contributes to the reperfusion injury of SEC after cold preservation of the liver. Therefore, the presence of effective antioxidants in the early reperfusion phase may be beneficial for liver graft integrity.

[Description of a special form of postprandial hyperlipidemia: possible cause of premature coronary heart disease]. / Beschreibung einer besonderen Form von post-prandialer Hyperlipidämie: mögliche Ursache einer frühzeitigen koronaren Herzkrankheit.

We have studied the metabolism of post-prandial lipoproteins in four male patients who suffered from premature (less than 60 years) angiographically proven coronary heart disease. They had normal to low total and low density lipoprotein cholesterol levels. Other risk factors were absent. Using the vitamin A fat loading test we were able to show that the clearance of chylomicron remnants was delayed compared to three healthy control subjects. Since these postprandial lipoproteins have been implicated in atherogenesis, we conclude that this defect contributed to the development of coronary heart disease in these patients.

Do intracellular, extracellular or urinary magnesium concentrations predict renal retention of magnesium in critically ill patients?

BACKGROUND AND OBJECTIVE: Magnesium disorders are common in hospitalized patients. In patients with low or normal magnesium, the intravenous magnesium loading test has been demonstrated to be a sensitive test to assess magnesium deficiency in critically ill patients. However, it is more time consuming and more difficult than the measurement of intracellular or extracellular magnesium concentrations. This study evaluated whether erythrocyte, plasma and urinary magnesium concentrations predict renal magnesium retention measured by th magnesium loading test. METHODS: One-hundred-and-three intensive care patients (36 females, 67 males) in a tertiary care centre and 41 healthy subjects (13 females, 28 males) took part in this prospective study. Intracellular, total plasma, ionize extracellular and urinary magnesium concentrations were measured and also magnesium retention by intravenous magnesium loading test. RESULTS: Total plasma magnesium concentration was poorly correlated with magnesium retention (r = 0.36 r2 = 0.13) and was the only parameter that significantly predicted magnesium retention in intensive care patients (P < 0.01). However, only 10% of the magnesium retention data were linked to the total plasma magnesium concentration. CONCLUSIONS: Total plasma magnesium concentration predicts magnesium retention in critically ill intensive care patients but not intracellular and urinary magnesium concentrations. Only a small proportion of the magnesium retention was due to the total plasma magnesium concentration.

The beneficial influence of nifedipine on the regression of the cholesterol-induced atherosclerosis in rabbits.

Nifedipine has been implicated as an inhibitor of dietary induced atherosclerosis in rabbits. This study was designed to examine the effect of nifedipine on the regression of atherosclerotic lesions in this animal model. NZ-rabbits were fed a cholesterol-enriched diet (1.5%) for 4.8 months. A control group A was killed and the aorta removed for planimetry of the vessel wall lesions. The remaining animals were divided into two groups, group P receiving a placebo solution and group N a nifedipine solution (2 X 20 mg/day). They were maintained on a standard chow for a further 4.5 months. In the nifedipine-treated group N, sudanophilia of the aorta was reduced by more than 20% as compared to the cholesterol-fed control group A and was 50% lower than in the placebo-treated group. Total cholesterol of the aortic tissue was lowest in group N. No significant differences in plasma cholesterol, triglycerides or the platelet half-life time were observed between the placebo- and the nifedipine-treated group. These data indicate that nifedipine can stimulate regression of pre-existing atherosclerotic lesions in rabbits.

Influence of bile acids and free fatty acids on physicochemical properties of LP-X.

In this study it is demonstrated, that incubation of both, bile acids and free fatty acids with LP-X, the abnormal plasmalipoprotein found in patients suffering from cholestasis or LCAT-deficiency, results in striking alterations of the physico-chemical and immunological properties of LP-X: 1. The cathodic mobility in agar is changed into an anodic mairation of the material. 2. The unique appearance of LP-X on electronmicrographs is altered by the incubation revealing fingerprint like structures. 3. The albumin portion of LP-X becomes immunologically detectable. 4. Bile salts cause marked changes in the hydrated density of the material as determined by zonal ultracentrifugation. 5. In vitro incubation of LP-X with postheparin plasma causes a complete disappearance of LP-X as judged by its typical migration on agar electrophoresis. All these alterations can be prevented or reversed by the addition of albumin in appropriate concentrations. These findinga are important in the light of studies designed to investigate the catabolic action of plasma lipolytic enzymes on LP-X, as well as for follow up studies of LP-X concentrations during the course of disease.

Determination of monoethylglycinexylidide by fluorescence polarization immunoassay in highly icteric serum samples: modified precipitation procedure and HPLC compared.

Hyperbilirubinemia, which frequently occurs in severe liver disease, interferes with the fluorescence polarization immunoassay (FPIA) monoethylglycinexylidide (MEGX) assay manufactured by Abbott Diagnostics. Because the MEGX test is particularly helpful in this clinical situation, strategies have been developed to overcome this problem. Precipitation of serum with the Abbott Digoxin II precipitation reagent eliminates bilirubin. Therefore, we compared FPIA results after precipitation of 81 icteric samples from 27 MEGX tests to results obtained using a validated HPLC method. The precipitation did not substantially alter the performance characteristics of FPIA: detection limit, 8 microg/L; between-days imprecision, 5.3-6.2%; recovery, 102-104% (50-200 microg/L). This pretreatment of serum did not eliminate all interference, and only a poor correlation was observed between serum MEGX concentrations measured with HPLC or modified FPIA (r2 = 0.46; S(y/x) = 20.0 microg/L). In contrast, MEGX formation values calculated by subtraction of the prelidocaine MEGX concentration were in close agreement (r2 = 0.98; S(y/x) = 2.3 microg/L). Because only MEGX formation is clinically relevant, this modified FPIA procedure offers a simple and rapid alternative to HPLC.

Critical evaluation of the 'heated-hand-technique' for obtaining 'arterialized' venous blood: incomplete arterialization and alterations in glucagon responses.

In order to test the degree of 'arterialization' and the occurrence of arterio- (or capillary-) venous differences in glucose concentrations for commonly used blood sampling sites (including the retrogradely cannulated dorsal hand vein with application of dry heat to this hand/arm--the 'heated-hand-technique'), oxygen partial pressure (oxygen saturation) and plasma glucose was determined in blood drawn from different venous sites before and after an oral glucose load (75 g). Experiments with and without heating (hot air 68 degrees C) were compared in nine healthy volunteers. Basal pO2 (and oxygen saturation) increased in the order cubital fossa vein less than superficial forearm vein less than dorsal hand vein. Heating raised pO2 by approximately 20 mmHg; P = 0.008) and oxygen saturation (P = 0.008-0.02) at all sites, including those on the contralateral arm. Capillary-venous glucose differences after the glucose challenge were significantly related to the sampling site (P less than 0.0001). They were reduced by approximately 50% in response to heat exposure (P = 0.008-0.011) and could be correlated to pO2-values (r = 0.92; P = 0.01). The lowest capillary-venous glucose concentration difference was measured with the 'heated-hand-technique' (0.4 +/- 0.1 mmol l-1). Heating did not alter integrated incremental glucose (capillary values), insulin, and C-peptide-responses and late, counter-regulatory responses (120-240 min after glucose) of cortisol, growth hormone, and adrenalin. However, the late glucagon response was enhanced (P = 0.011) by heating, concomitant with a significantly reduced 'reactive' decrement in glucose concentrations. In conclusion, the 'heated-hand-technique' provides blood more similar to arterial blood that can be obtained from other venous sampling sites. However, significant residual differences in pO2 and glucose concentrations remain. In addition, altered counter-regulatory hormone responses may occur with heating.

[Accuracy of HDL cholesterol measurements]. / Richtigkeit der HDL-Cholesterinmessung.

The widespread use of different methods for the determination of HDL-cholesterol (in Europe: sodium phosphotungstic acid/MgCl2) in connection with enzymatic procedures (in the USA: heparin/MnCl2 followed by the Liebermann-Burchard method) but common reference values makes it necessary to evaluate not only accuracy, specificity, and precision of the precipitation step but also of the subsequent cholesterol determination. A high ratio of serum vs. concentrated precipitation reagent (10:1 V/V) leads to the formation of variable amounts of delta-3.5-cholestadiene. This substance is not recognized by cholesterol oxidase but leads to an 1.6 times overestimation by the Liebermann-Burchard method. Therefore, errors in HDL-cholesterol determination should be considered and differences up to 30% may occur between HDL-cholesterol values determined by the different techniques (heparin/MnCl2 - Liebermann-Burchard and NaPW/MgCl2-CHOD-PAP).

Different modes of fragmenting gallstones in extracorporeal shockwave lithotripsy.

Forty radiolucent gallbladder stones from eight patients were fragmented in vitro by extracorporeal shockwave lithotripsy, using the electromagnetic lithotripter Lithostar Plus (Siemens) at five different energy levels. The stones were characterized by size, computed tomography (CT) density, and cholesterol content. The largest residual fragment was measured after every 20 to 100 shock waves. As expected, fewer shock waves were required to achieve fragmentation at higher energy levels. When stones of the same size were compared, there were remarkable differences in the number of shock waves required for fragmentation. These differences must originate in other properties of the stones than size and number. Two different modes of fragmentation were observed: in one group of stones small, flat fragments were chipped off at the beginning of fragmentation ('chipping mode'). These stones initially lost about 25% of their weight as small fragments (< 1 mm) before breaking centrally into some large fragments. In the other group stones initially lost only about 10% of their weight as small fragments (< 1 mm) at the beginning of fragmentation and early broke centrally into some large fragments ('breaking mode'). Stones showing the chipping mode were almost pure cholesterol stones (> 97%) and required significantly less shock waves than stones of the same size showing the breaking mode (cholesterol content, 64-94%). This mode of fragmentation could not be predicted by CT density.

Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit.

BACKGROUND: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated. METHODS: The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. RESULTS: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C. CONCLUSIONS: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.

[Defective remnant clearance as a risk factor for coronary heart disease]. / Gestörte Remnantclearance als Risikofaktor für die koronare Herzkrankheit.

We investigated the metabolism of postprandial lipoproteins in four male patients who suffered from premature (less than 60 years) angiographically proven coronary heart disease. They had normal to low plasma total and low density lipoprotein cholesterol levels. Other risk factors were absent. Using the vitamin A fat loading test we were able to show that the clearance of chylomicron remnants was delayed compared to three healthy control subjects. It has been suggested that these postprandial lipoproteins may be atherogenic. Our data strongly favour the concept that defective removal of postprandial lipoproteins leads to the development of coronary heart disease in these patients.

A simple and reliable reverse-phase high-performance liquid chromatographic procedure for determination of paclitaxel (taxol) in human serum.

A simple, fast, and reliable isocratic (mobile phase: acetonitrile/methanol/water [48/11/41], reverse-phase (C18 column) high-performance liquid chromatography method for the determination of paclitaxel concentration in human serum is presented. The procedure uses a new and convenient one-step sample-purification procedure that requires only 400 microliters of sample and uses N-heptylbenzamide as an internal standard. Paclitaxel is detected by UV absorbance measurement at 227 nm. The method has a broad linear range (0.01 to 10 mg/l, or 0.012 to 11.7 mumol/l; r > 0.999), and the detection limit is 0.01 mg/l (0.012 mumol/l). The deviation from target value is < or = 1.5%, and coefficients of variation are < or = 13.8% within runs and < or = 15.3% between runs. Recovery paclitaxel is > or = 92.6%. No interferences were observed from endogenous compounds or from more than 30 drugs that may be administered with paclitaxel. Docetaxel, which is not concurrently administered, coeluted with paclitaxel. Compared with previously published high-performance liquid chromatography procedures for the determination of paclitaxel, the particular advantage of the method presented here is its simple and rapid single-step sample-purification procedure, which makes a high recovery of paclitaxel from serum samples possible and results in a pure extract, avoiding interferences from endogenous compounds. The method is suitable for pharmacological studies and routine analysis.