A NMR-compatible and reduced gravity simulation based (NRG) bioreactor for on-line monitoring cell culture metabolism.

We developed a NMR-compatible microgravity-based bioreactor (NRG[R]) that offers the advantage of an analytical non-invasive approach associated to the effects of an optimized suspension culture. The simulated microgravity conditions reached in the bioreactor are analogous to those of commercial apparatus like the Rotating Wall Vessel (RWV) system. The faster proliferation of endothelial cells cultured in the NRG bioreactor (doubling time : 28 +/- 1.7 vs. 43 +/- 5.6 h of the control grown in RWV) are attributed to different oxygenation conditions and medium wash out.

Glycogen turnover and anaplerosis in preconditioned rat hearts.

Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.

Does resveratrol induce pharmacological preconditioning?

Resveratrol is a grape component with complex pharmacology related to its antioxidant activity. Little is known about the direct effects of resveratrol on the myocardium. We tested whether resveratrol administration before ischemia could attenuate ischemic/reperfusion damage. We examined how resveratrol affects high-energy phosphate metabolism (31P-nuclear magnetic resonance) and contractility of isolated Langendorff perfused rat hearts subjected to 20 min no-flow ischemia and 30 min reperfusion. During 10 min resveratrol infusion (10 microM) before ischemia, basal phosphorylation potential dropped by 40% (p < 0.05 vs. preinfusion value) without affecting contractility. The level of effluent adenosine was increased by 68%, parallel to a 50% increase in coronary flow. Resveratrol significantly improved postischemic recovery of rate-pressure product (62 +/- 5.2 vs. 23 +/- 8.1% of controls; p < 0.05). The metabolic pattern following resveratrol infusion was similar to that produced by ischemic preconditioning, suggesting that an increase in adenosine availability is involved in cardioprotection.

Induction of human endothelial cell growth by mildly oxidized low density lipoprotein.

The aim of the present study was to examine whether endothelial growth could be modulated by mildly oxidized low density lipoprotein. When human endothelial cells were cultured in the presence of mildly oxidized low density lipoprotein (1 microgram/ml), a significant induction of endothelial cell growth was observed, whereas native low or high density lipoprotein were ineffective. Further, treatment of endothelial cells with mildly oxidized low density lipoprotein modulated the expression of cytokines and growth factors which may be relevant in atherogenesis. Endothelial cells chronically exposed to mildly oxidized low density lipoprotein underwent a more rapid onset of cellular senescence. Since senescence is associated with endothelial dysfunction, the novel finding showing that mildly oxidized low density lipoprotein induces endothelial cell growth may be relevant in the development and evolution of the atherosclerotic lesions.

Modulators of oxidized LDL-induced hyperadhesiveness in human endothelial cells.

The adherence of monocytes to the endothelium is an early event in atherogenesis. Our previous studies have demonstrated that oxidized LDL induced U937 cells-endothelial interactions and that HDL prevented oxidized LDL effects. Here, we provide evidence that treatment of endothelial cells with the anti-inflammatory agent indomethacin abolished oxidized LDL as well as interleukin 1- and lipopolysaccharide-stimulated U937 adhesion. It is noteworthy that HDL, which is known to be protective against atherosclerosis, was effective only in negating U937 adhesion induced by oxidized LDL, while it did not affect interleukin 1- and lipopolysaccharide-induced hyperadhesiveness in endothelial cells. Since indomethacin inhibits cyclooxygenase which is the key enzyme in the synthesis of prostanoids, we have studied the effect of oxidized LDL on the expression of cyclooxygenase type 2 and demonstrated that oxidized LDL induces a sustained increase in the expression of cyclooxygenase mRNA.

The protective role of high-density lipoprotein on oxidized-low-density-lipoprotein-induced U937/endothelial cell interactions.

The adherence of monocytes to the endothelium is an early event in atherogenesis. We have investigated this process by examining whether native and oxidized low-density and high-density lipoproteins could modulate this process. Only oxidized low-density lipoprotein caused a significant dose-dependent and time-dependent increase in U937 monocyte-like cell line binding to human endothelial cells, by a process which required de novo protein synthesis. Interestingly, E-selectin, intercellular adhesion molecule-1, vascular cell-adhesion molecule or P-selectin induction was not apparent in this system suggesting the presence of an alternative system for the interaction of endothelial cells with monocyte-like cells in response to oxidized low-density lipoprotein. High-density lipoprotein completely suppressed oxidized low-density-lipoprotein-induced adhesion of U937 cells to the endothelial monolayer, while oxidized high-density lipoprotein did not. These data suggest that the balance between native and oxidized lipoproteins may play a role in the formation of the atherosclerotic lesion by modulating monocyte endothelial interactions.

Free radicals promote modifications in plasma high-density lipoprotein: nuclear magnetic resonance analysis.

The behavior of high-density lipoprotein (HDL) after free-radical-mediated oxidation was studied by incubating plasma HDL with chemical oxidizing systems (Cu++) in conditions similar to those used for low-density lipoprotein (LDL) chemical oxidation. Nuclear magnetic resonance (NMR) spectroscopy (1H and 31P) was used to evaluate the degree of oxidation and to characterize the oxidized products. The almost complete loss of polyunsaturated systems together with an appreciable decrease in choline peak demonstrates large-scale HDL-lipid degradation. The appearance of epoxide systems on fatty chains and the identification of oxidized cholesterol derivatives as cholesterol 5 alpha,6 alpha-epoxide, 5 beta,6 beta-epoxide, 7-keto, and 25-hydroxy confirm this picture. Phospholipid analysis indicates an alteration of the phospholipid profile in lyso-phosphatidylcholine (Lyso-PC) production and the disappearance of phosphatidylethanolamine (PE). This study shows that HDL is extensively degraded although there are no large variations in the classical oxidative monitors, lipid hydroperoxide (LPO) and thiobarbituric acid reactive substance (TBARS). Our results suggest that HDL is significantly modified when submitted to an oxidative process.

Human apolipoprotein A-I gene promoter polymorphism: association with hyperalphalipoproteinemia.

An apolipoprotein A-I gene promoter polymorphism, due to an adenine (A) to guanine (G) transition 78 base pairs upstream from the transcription initiation site, was studied by amplification of the corresponding region of the apoA-I gene, DNA sequencing, and allele-specific oligonucleotide hybridization. The frequency of the polymorphism was studied on female and male individuals classified into three groups according to the high density lipoprotein (HDL) cholesterol concentration. The allelic frequencies for the A polymorphism were 0.10, 0.14, 0.27 in women and 0.08, 0.17, 0.14 in men in the lowest, in the intermediate (between 10th and 90th percentile), and the highest decile of HDL cholesterol levels, respectively. Statistical analysis showed a significant difference of allelic frequencies between the highest and the lowest deciles (P less than 0.006) and between the highest and the intermediate deciles of HDL cholesterol in women (P less than 0.04) but not in men. As the sequences surrounding the polymorphism are known to be involved in transcription modulation, it is possible that the A-G transition polymorphism may have an influence on apoA-I synthesis and, in consequence, on the HDL cholesterol levels in women.

An alternative expeditious analysis of phospholipid composition in human blood plasma by 31P NMR spectroscopy.

31P nuclear magnetic resonance spectroscopy has been applied to quantitate phospholipids in human blood plasma and in separate lipoprotein fractions. The addition of suitable detergents to samples produces an excellent chemical shift dispersion and allows the identification and integration of the peaks of the most important phospholipids. Results are in agreement with those obtained with enzymatic colorimetric and TLC methods: our method is characterized by good accuracy and reproducibility.

NMR analysis of low-density lipoprotein oxidatively-modified in vitro.

Human plasma low density lipoprotein has been oxidized at different stages in vitro and analysed by 1H, 13C, and 31P NMR spectroscopy and by biochemical methods. Information was obtained on: a) structure mobilities of lipids and on lipid-protein interactions; b) conjugated and oxo-dienes; c) polyunsaturated/monounsaturated fatty acid chains; d) lysophosphatidylcholine production. The results show that the NMR approach is particularly useful for the assessment of structural modification in oxidized LDL.

Erythrocytic glucose-6-phosphate dehydrogenase measured by a differential pH technique.

We propose a new quantitative electrochemical method for determining glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in purified erythrocytes or in whole blood, based on measurement of the pH change caused by oxidation of glucose 6-phosphate to 6-phosphogluconic acid, with simultaneous reduction of NADP+ to NADPH + H+. No sample pretreatment (e.g., preparation of hemolysate) is needed, and the automatic correction for sample blanks obviates interference from 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The method is simple and fast, and the standard curve is linear to at least 2200 U/L at 37 degrees C. The within-day CV was 3.9% for activities in healthy individuals (mean value 1204 U/L), and 10% for deficient ones (classified as belonging to class II, mean value 407 U/L). Results (y) correlated well with those obtained with the WHO-recommended method (x): y = 1.13x + 0.02 (r = 0.971) for purified erythrocytes. Normal reference intervals are reported for the enzyme in purified erythrocytes and in whole blood.

Effects of suprofen on renal function in patients with rheumatoid arthritis.

Suprofen is a new potent analgesic with antiinflammatory properties that appears to inhibit prostaglandin synthetase in a tissue-selective manner, having relatively little effect on the kidneys of experimental animals. The effects were studied of one week of treatment of rheumatoid arthritis patients with suprofen or ibuprofen on Na+ and K+ excretion, creatinine clearance, urinary enzymes that are markers for tubular damage, and urinary prostaglandins such as PGE2 and 6-keto PGF1 alpha (a stable metabolite of prostacyclin). Neither compound caused changes in renal function related to the week of treatment, but significant decreases in prostaglandins were observed: this change was fully reversible after discontinuation of the drug.

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30 degrees C and correlate well with the manual spectrophotometric method of Ellman for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

Optimization of the urinary acid alpha-glucosidase determination.

The optimization of the method for acid alpha-glucosidase determination (EC 3.2.1.3) in human urines, employing the synthetic substrate 4-nitrophenyl-alpha-D-glucopyranoside, is reported. Storage conditions of the specimens and their pretreatment were particularly investigated. The precision of the whole analytical procedure (including gel filtration) is good (within-run CV = 7.4% for normal samples and 3.7% for elevated ones). The correlation with the method using maltose as substrate is excellent (y = -0.01 + 0.13 x; r = 0.9893).

Measurement of lipase activity by a differential pH technique.

This is a new electrochemical method for determination of lipase activity in biological fluids, including serum, plasma, and duodenal juice. Advantages of turbidimetric methods--short reaction time, and small sample and reagent volumes--are combined with those of titrimetric methods: measurement of absolute activity (i.e., no standardization required), saturated substrate conditions, and direct measurement of reaction products. The proposed method is easy, inexpensive, and takes only 3 min. Precision is good: CV = 3.74% within day and 7.3% between days at the clinical-decision concentration, CV = 1.86% within day and 4.65% between days for above-normal lipase activities. The standard curve is linear up to 4500 U/L. Results (y) correlate well with those by turbidimetry (x): y = 0.9287x - 65.3 (r = 0.9719). Reference values are between 0 and 130 U/L.

Fusion of sulfatide-containing vesicles of phosphatidylcholine.

Positively charged albumin is described as a 'useful tool' to induce both aggregation and fusion of phosphatidylcholine vesicles containing sulfatide. Techniques that include light-scattering, Sepharose chromatography, centrifugation, electron microscopy, trapped volume determination and scanning calorimetry demonstrate that extensive fusion occurs during aggregation when sulfatide concentrations are above 4-5 mol%. The rate of fusion increases with time for 1-2 h, then reaches a plateau. Fusion occurs extensively above the transition temperature of the phospholipid and is strongly inhibited by increasing concentration of vesicle cholesterol. The significance of both membrane fluidity and sulfatide-phospholipid organization in the fusion mechanism are discussed.