Decreased erythrocyte membrane fluidity in poorly controlled IDDM. Influence of ketone bodies.

OBJECTIVE: To examine the factors that might alter the fluidity of erythrocyte membrane in insulin-dependent diabetes mellitus (IDDM) patients. RESEARCH DESIGN AND METHODS: The subjects were 10 health men and 30 IDDM mem: 10 with good blood glucose (BG) control (HbA1c 5.88 +/- 0.60% [mean +/- SD]), 10 with poor BG control (HbA1C 9.48 +/- 1.05%), and 10 with poor BG control and mild to moderate diabetic ketoacidosis (DKA) (HbA1C 9.12 +/-2.25%, strongly positive ketonuria 3+ and elevated plasma beta-hydroxybutyrate). Erythrocyte membrane fluidity was determined by fluorescence polarization using 6-(9-anthroyloxy stearic acid as fluorescent probe. RESULTS: Membrane fluidity was normal in the diabetic patients with good BG control but significantly lower in the two groups of patients with poor BG control than in the healthy subjects (P < 0.01). The membrane fluidity in the poor BG control groups was also lower in the patients with DKA than in those without DKA (P < 0.01). CONCLUSIONS: The factors that most influence membrane fluidity in IDDM patients appear to be hyperglycemia and ketone bodies.

Hyperinsulinemia is related to erythrocyte phospholipid composition and membrane fluidity changes in obese nondiabetic women.

It has been suggested that changes in the properties of cell membranes are involved in an altered insulin action. However, the influence of changes in the distribution of phospholipid classes has not been explored. We investigated 69 obese nondiabetic normoglycemic women (17 patients with impaired glucose tolerance) with varying degrees of insulin sensitivity to determine the phospholipid composition and fluid state of their erythrocyte plasma membranes. The fasting plasma insulin, the homeostasis model analysis of insulin resistance (HOMA), and the integrated area under the insulin curve (AUC-I) after an oral glucose challenge were used as markers of insulin resistance. Results were divided into normal glucose tolerance (NGT) and impaired glucose tolerance. There was a positive correlation in NGT group between the membrane sphingomyelin (SM) content and the fasting plasma insulin (r = 0.523; P < 0.0001), HOMA value (r = 0.483; P < 0.0005), and AUC-I (r = 0.352; P < 0.05) and negative correlations between membrane fluidity determined with two fluorescent probes and plasma fasting insulin (r = 0.320; r = -0.365; P < 0.05) and HOMA value (r = 0.321; r = -0.382; P < 0.05). There were also correlations between SM and the three markers of insulin resistance in the impaired glucose tolerance group. There was no correlation between insulin resistance and other membrane components. Stepwise multiple regression analysis in the NGT group confirmed that the membrane SM content was an independent predictor of plasma fasting insulin, HOMA values, and AUC-I variations. Sphingomyelin could be one of the membrane parameters contributing to insulin resistance.

MTT assays allow quick and reliable measurement of the response of human tumour cells to photodynamic therapy.

MCF-7 and HT-29 cell lines were selected as a reliable model to examine the possible parameters affecting the sensitivity of tumour cells to photodynamic therapy (PDT) using a dye-laser at 630 nm. The chemical composition of haematoporphyrin derivative (HPD) was determined by high-performance liquid chromatography (HPLC) analysis and was in agreement with reported values. MTT assays were performed to assess the time-dependency of PDT and the influence of the output power and light fluence. The results showed a maximal cytotoxicity 48 h after photoirradiation. The output power (1 or 2 W) did not significantly affect the cytotoxicity when the fluence was constant (20 J/cm2). However, an increase in fluence (10-40 J/cm2) led to a significant enhancement of cytotoxicity until maximal values were reached (30-40 J/cm2). A further increase in fluence (50 J/cm2) proved to induce a fall-off in cytotoxicity related to the intense photobleaching of HPD.

Effects of lipid supplementation of culture media on cell growth, antibody production, membrane structure and dynamics in two hybridomas.

Membrane lipid organization and membrane fluidity affect cell functions. The effects of supplementing culture media with a lipid mixture (Ex-Cyte) containing cholesterol, phospholipids and fatty acids on cell growth, antibody production and membrane composition and dynamics in two hybridoma cell lines were studied. A49 cells decreased immunoglobulin production but cell growth increased. Lipids had no effect on the cell growth rate of B9 cells but increased immunoglobulin production and productivity. The fluidity of the deep areas of the plasma membrane and cytoplasmic organelles increased in the two cell lines. There was increased fluidity of the polar regions of the plasma membrane and a decreased phosphatidylethanolamine/phosphatidylcholine ratio in A49 cells. B9 cells underwent no change in fluidity of the polar regions but the phosphatidylinositol content was increased, together with higher monoclonal antibody production. These results demonstrate that antibody production is not linked to the dynamic properties of the membrane, even though changes in the membrane phosphatidylinositol content are associated with the final step of antibody secretion, but that the action of phospholipids and fatty acids on cell growth is membrane-associated.

Spectroscopic and biological testing of photobleaching of porphyrins in solutions.

The photobleaching of protoporhyrin IX (PP IX) and hematoporphyrin derivative (HpD) solutions was followed using three different methods: spectrophotometry, fluorometry and photodynamically induced cytotoxicity. The latter entails photoirradiation of HT29 human colon adenocarcinoma cells in the presence of preirradiated solutions of HpD and PP IX (lambda < or = 415 nm). The highest cytotoxicity was observed in the presence of unirradiated dye and decreased with the time of preirradiation. This decay in photocytotoxicity was further used to determine the porphyrin photobleaching kinetics in solution. For both sensitizers, quantum yields of photobleaching obtained by matching fluorescence were higher than that obtained from absorbance measurements (10 and 11 times for HpD and PP IX, respectively). This difference reflects preferential photobleaching of photolabile monomeric forms compared to aggregated. The highest quantum yield was obtained in the biological test (decay in cytotoxicity) which was 14 times higher for HpD and 30 times higher for PP IX than the quantum yield obtained from absorbance measurements. The absence of correlation between biological and fluorescence measurements has to be taken into account in the in vivo situation. Dark storage of preirradiated sensitizers (37 degrees C, 24 h) completely restored photocytotoxity for PP IX but only partially for HpD, whereas fluorescence patterns were partially restored for both sensitizers.

The IPSO study: ibuprofen, paracetamol study in osteoarthritis. A randomised comparative clinical study comparing the efficacy and safety of ibuprofen and paracetamol analgesic treatment of osteoarthritis of the knee or hip.

OBJECTIVE: To compare the analgesic efficacy of single and multiple doses of ibuprofen with that of paracetamol in patients with knee or hip osteoarthritis (IPSO study). METHOD: 222 patients were randomised in a double blind, multicentre study-156 (70%) had a painful knee joint and 66 (30%) a painful hip joint. The main efficacy criterion was pain intensity assessment after a single dose (ibuprofen 400 mg, paracetamol 1000 mg). Functional disability assessment and patient global assessment were carried out over 14 days. RESULTS: The sum of the pain intensity difference over 6 hours after the first administration was significantly higher (p = 0.046) in the ibuprofen group than in the paracetamol group. Over 14 days pain intensity decreased from the first day and was significantly lower in the ibuprofen group than in the paracetamol group (p<0.05). The functional disability of the patient was assessed using the WOMAC; the ibuprofen group improved significantly over 2 weeks compared with the paracetamol group for each of the subscales: stiffness (p<0.002), pain (p<0.001), physical function (p<0.002). The drugs were equally safe. CONCLUSION: The IPSO study shows that for the treatment of osteoarthritic pain, ibuprofen 400 mg at a single and multiple dose (1200 mg/day) for 14 days is more effective than paracetamol, either as a single dose of 1000 mg or a multiple dose (3000 mg/day). Because ibuprofen and paracetamol have similar tolerability, this study indicates that the efficacy/tolerability ratio of ibuprofen is better than that of paracetamol in this indication over 14 days.

Adipocyte and erythrocyte plasma membrane phospholipid composition and hyperinsulinemia: a study in nondiabetic and diabetic obese women.

BACKGROUND: The cell functions involved in the action of insulin--receptor binding, enzyme and transporter activities--are controlled by membrane properties. We have previously shown that the fasting plasma insulin (FPI) concentration and the homeostasis model assessment (HOMA) estimate of insulin resistance are associated with the sphingomyelin concentration in the erythrocyte membranes of obese women. OBJECTIVES: (1) To study the distribution of phospholipid classes in the plasma membrane and their association with insulin resistance markers in the adipocyte, an insulin-sensitive cell in obese women. (2) To investigate the influence of diabetes in a small group of obese women treated by diet alone. (3) To compare the distribution of phospholipids in erythrocyte membranes in a subgroup of obese nondiabetic and diabetic women. SUBJECTS: Subcutaneous fat biopsies were taken from the abdominal region of 19 obese non-diabetic and seven obese type 2 diabetic women. Erythrocyte membrane assessment was performed in a subgroup of 10 of the 19 obese nondiabetic and in the seven diabetic patients. METHODS: The phospholipid composition of adipocyte and erythrocyte plasma membranes was analyzed by high performance liquid chromatography. RESULTS: FPI was positively correlated with the adipocyte membrane contents of sphingomyelin (P < 0.001), phosphatidylethanolamine (P < 0.05), and phosphatidylcholine (P < 0.01) in the obese nondiabetic women. Similar correlations were obtained with HOMA. A stepwise multiple regression analysis indicated that sphingomyelin accounted for 45.6 and 43.8% of the variance in FPI and HOMA values as an independent predictor. There was a similar positive independent association between FPI and SM in the erythrocyte membranes of the studied subgroup. Diabetes per se did not influence the independent association between SM membrane contents and FPI in both cell types. CONCLUSION: These results suggest a link between membrane phospholipid composition, especially SM, and hyperinsulinemia in obese women.

Adipocyte membrane phospholipids and PPAR-gamma expression in obese women: relationship to hyperinsulinemia.

We have shown that membrane sphingomyelin (SM) is an independent predictor of the variance of fasting plasma insulin (FPI) concentrations and the homeostasis model assessment (HOMA) estimate of insulin resistance in obese women. The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a key component in adipocyte differentiation that may also contribute to the sensitivity of cells to insulin. PPAR-gamma is activated by fatty acids, and the membrane composition may have an impact on the activity of PPAR-gamma and thus on the sensitivity of adipocytes to insulin. We investigated these possible links by determining the phospholipid contents of adipocyte membranes, the mRNA expression of PPAR-gamma, and the FPI and HOMA estimate of insulin resistance in obese women. The mRNA levels of tumor necrosis factor-alpha (TNF-alpha), which is suspected to play a role in insulin resistance and which downregulates PPAR-gamma expression, were also quantified. FPI and HOMA were strongly positively correlated with membrane SM (P < 0.005) and cholesterol (P < 0.005). PPAR-gamma mRNA levels were negatively correlated with FPI (P < 0.05) and HOMA (P < 0.05) and positively correlated with high-density lipoprotein (HDL) cholesterol (P < 0.05), membrane SM (P < 0.05), and cholesterol contents (P < 0.05). TNF-alpha mRNA levels were not correlated with membrane parameters. In stepwise multiple regression analysis, the variations in PPAR-gamma mRNA levels were mainly explained by HDL cholesterol (31.9%) and membrane SM (17.7%). Our study shows that the expression of PPAR-gamma, a major factor controlling adipocyte functions, the lipid composition of the membrane, and insulin sensitivity are probably closely associated in the adipose tissue of obese women.