A 26-week, placebo- and pioglitazone-controlled monotherapy study of rivoglitazone in subjects with type 2 diabetes mellitus.

AIMS: To evaluate the efficacy and safety of rivoglitazone, a peroxisome proliferator-activated receptor γ agonist in the thiazolidinedione class, in subjects with suboptimally controlled type 2 diabetes mellitus (T2DM). METHODS: Subjects aged ≥18 years with T2DM and haemoglobin A1c (HbA1c) >7.0% and ≤8.5%, who were treatment naïve or receiving a non-thiazolidinedione antidiabetes monotherapy, entered a 2-week washout and single-blind placebo run-in period and were then randomized 2 : 4 : 11 : 11 to double-blind treatment with placebo, rivoglitazone 1.0 mg/day, rivoglitazone 1.5 mg/day, or pioglitazone 45 mg/day, for 26 weeks. RESULTS: A total of 1912 subjects received placebo (n = 137), rivoglitazone 1.0 mg (n = 274), rivoglitazone 1.5 mg (n = 750) or pioglitazone (n = 751). Rivoglitazone 1.5 mg was statistically superior (p = 0.0339) and rivoglitazone 1.0 mg was non-inferior (p = 0.0339) to pioglitazone in reducing HbA1c from baseline (changes of -0.7%, -0.4% and -0.6%, respectively). Rivoglitazone also significantly reduced fasting plasma glucose from baseline (p < 0.0001). Rivoglitazone significantly improved estimates of insulin sensitivity, high-density lipoprotein cholesterol levels, and other metabolic and inflammatory biomarkers. Rivoglitazone was generally well tolerated at both doses, with treatment-emergent adverse event (TEAE) rates similar to pioglitazone. The most common drug-related TEAEs were peripheral oedema (active, 5.2-6.2%; placebo 0.7%), increased weight (active, 1.6-3.1%; placebo, 0%) and pitting oedema (active, 1.3-2.2%; placebo, 0%). CONCLUSIONS: In subjects with suboptimally controlled T2DM, rivoglitazone 1.5 mg was associated with statistically superior glycaemic control to pioglitazone 45 mg, while rivoglitazone 1.0 mg was non-inferior; the safety profiles of the two drugs appeared similar.

Dialysis solution containing hyaluronan: effect on peritoneal permeability and inflammation in rats.

BACKGROUND: Hyaluronan (HA), a high molecular weight mucopolysaccharide found in interstitial tissues and fluid, is lost from the peritoneal cavity during peritoneal dialysis. In order to determine the role of HA in peritoneal function, we investigated the effects of exogenous HA on peritoneal permeability, markers of intraperitoneal inflammation, and peritoneal morphology in rats exposed to peritoneal dialysis solution for four weeks. METHODS: Wistar rats were infused intraperitoneally, twice daily, with conventional, hypertonic dialysis solution (Dianeal 3.86%; control) or Dianeal solution containing 10 mg/dL of high molecular weight HA. Peritoneal permeabilities and clearances of solutes and protein were determined using a modified peritoneal permeability test (peritoneal equilibration test) at the beginning and the end of the treatment. Peritoneal volume and ultrafiltration were determined using a macromolecular marker and by gravimetric methods. Peritoneal inflammation was determined by cell counts and differential and by the measurement of cytokine concentrations in the dialysate effluent. Peritoneal thickness and HA content were determined in liver and mesentery biopsies taken at the end of the experiment. RESULTS: After four weeks of exposure to the dialysis solution, transperitoneal protein equilibration was significantly lower in HA-treated rats compared with rats treated with Dianeal alone (46% lower for albumin, P < 0.003; 33% lower for total protein, P < 0.001). The total drained volume after a four hour dwell was 29% higher in the HA group compared with the control (P < 0.001), yielding a positive net ultrafiltration in the HA group versus a negative net ultrafiltration in controls. Peritoneal clearances of urea and creatinine tended to be elevated in HA-treated rats, while clearances of total protein and albumin tended to be lower. Dialysate effluent from rats exposed to HA contained a lower percentage of neutrophils (8.8 +/- 22.8 +/- 9.5%, P < 0.01) and lower levels of the cytokines, tumor necrosis factor-alpha (11.2 +/- 14.7 vs. 42.3 +/- 35.3 pg/mL, P < 0.05) and monocyte chemoattractant protein-1 MCP-1 (72.0 +/- 86.5 vs. 402.4 +/- 258.3 pg/mL, P < 0.02), compared with rats treated with Dianeal alone. The thickness of the peritoneal interstitium showed a similar increase in both groups, but mesenteric tissue from the HA group contained more HA (48%, P < 0.01) than tissue from control animals. CONCLUSIONS: The addition of HA to peritoneal dialysis solution decreases protein permeability, increases ultrafiltration, and decreases cytokine levels and the proportion of peritoneal neutrophils in dialysate from rats exposed to hypertonic dialysis solution. These results suggest that exogenous HA may help to protect the peritoneal membrane during exposure to dialysis solutions. These benefits, if sustained in the clinical setting, could lead to improvements in the therapy of peritoneal dialysis.

l-2-oxothiazolidine-4-carboxylic acid modulates function of peritoneal mesothelial cells in vitro.

The influence of the glutathione precursor, l-2-oxothiazolidine-4-carboxylic acid (OTZ), on the function of human peritoneal mesothelial cells in vitro, in conditions that mimic the in vivo effect of peritoneal dialysis solutions on mesothelium, was studied. Mesothelial monolayers were exposed to dialysis fluids (Dianeal 1.36 or Dianeal 3.86; Baxter Healthcare Corp, Round Lake, IL) that were diluted gradually with pooled-effluent dialysate obtained from patients undergoing continuous ambulatory peritoneal dialysis. In vitro exposure of mesothelium to standard dialysis fluid enhances their susceptibility to injury by hydrogen peroxide. OTZ added to dialysis solution in concentrations of 1 mmol/L prevented the toxic effect of hydrogen peroxide, probably by increasing intracellular glutathione. Mesothelial cells exposed to dialysis fluid become activated, evidenced by increased release of interleukin-6 and hyaluronan. OTZ used in concentrations of 1 mmol/L reduced that effect. Furthermore, the addition of glucose to the culture medium in a concentration of 45 mmol/L inhibits the proliferation of mesothelial cells; the presence of OTZ, 1 mmol/L, partially prevents the inhibitory effect of glucose. The results presented in this report show that by augmenting the intracellular concentration of glutathione in mesothelial cells by the addition of OTZ to the dialysis fluid, we can increase their resistance to the acute toxicity of free radicals and long-term toxicity of glucose. In addition, mesothelial cells with an increased glutathione level are less activated after their exposure to dialysis fluid.

Effects of peritoneal rest on peritoneal transport and peritoneal membrane thickening in continuous ambulatory peritoneal dialysis rats.

OBJECTIVE: To evaluate the effects of peritoneal rest on peritoneal transport and morphology in a rat model of peritoneal dialysis. DESIGN: Twenty-four rats (Sprague-Dawley, male, 250-300 g) were divided into three groups: group 1 (control, n = 6) without dialysis, group 2 (n = 9) sacrificed immediately after 3 weeks of dialysis, and group 3 (n = 9) sacrificed after 4 weeks of peritoneal rest after 3 weeks of dialysis. Both dialysis groups were dialyzed twice daily with an intraperitoneal instillation volume of 25 mL of 3.86% dextrose solution for 3 weeks. Peritonitis was induced by supplementing the dialysis fluid with lipopolysaccharide (5 microg/mL) on days 8, 10, and 12 in both dialysis groups. Peritoneal equilibration tests were performed on each animal at baseline. The equilibration tests were repeated at the 4th and the 8th week of dialysis. Morphometric analyses of the peritoneal membrane were carried out in tissue specimens obtained at the time of sacrifice. RESULTS: The D/D0 ratio for glucose at two hours in groups 2 and 3 at the beginning of week 4 was significantly lower than at baseline, indicating an increase in peritoneal permeability to glucose after 3 weeks of dialysis. D/D0 in group 3 at the beginning of week 8, after 4 weeks of peritoneal rest, was significantly higher than at week 4. The drain volume in groups 2 and 3 at week 4 was significantly lower than at baseline; however, the drain volume in group 3 at week 8 was significantly higher than at week 4. The thickness of the parietal peritoneal membrane in group 3 was significantly greater than in group 1 and less than in group 2 (group 1, 11.4+/-7.6 microm; group 2, 37.5+/-18.4 microm; group 3, 21.4+/-12.1 microm). CONCLUSIONS: Peritoneal rest improves ultrafiltration in rats by decreasing the hyperpermeability of glucose and also reduces the degree of peritoneal thickening. These data suggest that dialysis-induced changes in peritoneal transport and morphology are reversible under the conditions of peritoneal rest in this experimental model.

Decreased in vitro formation of AGEs with extraneal solution compared to dextrose-containing peritoneal dialysis solutions.

Extraneal peritoneal dialysis (PD) solution (Baxter Healthcare, Deerfield, Illinois, U.S.A.) contains glucose polymer (icodextrin) as an osmotic agent in place of dextrose. We investigated the ability of Extraneal to form advanced glycation end products (AGEs) in vitro compared to standard PD solutions containing dextrose. Extraneal, Dianeal PD-2 [1.5%, 2.5%, or 4.25% dextrose (Baxter Healthcare)], or phosphate buffered saline (PBS) were incubated for 45 days with human serum albumin (HSA) or type IV collagen. AGE formation was measured by spectrofluorometry using excitation at 350 nm and emission at 430 nm. Solutions were also incubated with collagen affixed to plastic, simulating matrix collagen in the peritoneal membrane. In addition, AGE formation was assessed using icodextrin metabolites (maltose, maltotriose, and maltotetraose) at concentrations normally found in the plasma of patients treated using icodextrin. For PD solutions incubated with albumin, the relative order of AGE formation was: 4.25% dextrose > 2.5% dextrose > 1.5% dextrose > Extraneal. For incubations with collagen (in solution or affixed to plastic), AGE formation was greatest for 4.25% dextrose, intermediate for Extraneal and 2.5% dextrose, and lowest for 1.5% dextrose. Incubation of icodextrin metabolites with albumin for 45 days did not result in appreciable AGE formation. These results confirm that solutions containing icodextrin result in less in vitro AGE formation than do high dextrose solutions. The results also suggest that Extraneal may lead to improved solution biocompatibility in vivo.

Lack of interference of icodextrin on creatinine measurements.

Glucose has been reported to interfere in the analysis of creatinine by the Jaffe method. The potential interference of icodextrin and its primary metabolites (maltose, maltotriose, maltotetraose) on creatinine measurements has not previously been addressed. We evaluated the potential interference of icodextrin and its metabolites at various concentrations using both the Jaffe and Creatinine Plus methods. Interference was determined in samples containing 0.6-20 mg/dL creatinine in saline solution or in plasma (n = 6), and in dialysate samples (n = 6) spiked with icodextrin, maltose, maltotriose, and maltotetraose at concentrations up to twofold the level found in plasma and dialysate from patients treated using icodextrin. Results confirm that no interference occurs when using either the colorimetric Jaffe method or the enzymatic Creatinine Plus method at levels up to 65 g/L icodextrin, 2 g/L maltose, 2 g/L maltotriose, and 1 g/L maltotetraose, levels representing worst-case clinical concentrations. In addition, our results confirm that comparable values can be obtained using either the Jaffe or the Creatinine Plus method for the analysis of creatinine in uremic plasma and in dialysate samples.

Apolipoprotein C-III, hypertriglyceridemia and triglyceride-rich lipoproteins in uremia.

Apolipoprotein C-III (ApoC-III) plays an important role in the metabolism of triglyceride-rich lipoproteins and is known to be elevated in patients with uremia. To investigate the role of apoC-III in uremic dyslipidemia, we examined apoC-III, triglyceride levels and lipoprotein particles containing both apoB and apoC-III (LP-Bc) in 27 uremic patients prior to dialysis (predialysis), 30 patients on hemodialysis (HD) and 31 patients on peritoneal dialysis (PD). All three groups of patients had elevated levels of plasma apoC-III (20+/-7 mg/dl for predialysis, 18+/-5 for HD and 22+/-8 for PD, compared to 11+/-3 mg/dl for control subjects [p<0/01 for all comparisons]). ApoC-III was positively correlated with plasma triglycerides in PD patients (r = 0.86, p<0.0001), HD patients (r = 0.67, p<0.0001) and predialysis patients (r = 0.60, p<0.001) as well as in all patients combined (r = 0.75, p<0.0001). ApoC-III was also positively correlated with levels of LP-Bc in all three groups of patients, although this correlation was less strong (r = 0.46, p<0.0001 for all patients combined). In predialysis and PD patients, the majority of apoC-III was found in heparin precipitable lipoproteins, whereas the majority of apoC-III in HD patients was found in HDL, indicating less efficient lipolysis in predialysis and PD patients in comparison with HD. These data support the hypothesis that the elevation of apoC-III in uremia can alter the metabolism of triglyceride-rich lipoproteins, leading to an elevation in triglycerides and LP-Bc. Understanding the mechanism(s) of elevated apoC-III in uremia may help to clarify the causes of uremic dyslipidemia.

Elevation of whole-blood glutathione in peritoneal dialysis patients by L-2-oxothiazolidine-4-carboxylate, a cysteine prodrug (Procysteine).

Glutathione is a major cellular antioxidant that protects protein thiols and inhibits cellular damage due to oxygen free radicals. It has been reported previously that patients undergoing dialysis have low levels of blood glutathione, which may lead to increased susceptibility to oxidant stress. L-2-oxothiazolidine-4-carboxylic acid (OTZ) is a cysteine prodrug that raises cellular glutathione levels by increasing delivery of cysteine, the rate-limiting substrate for glutathione synthesis. This study investigates the effect of OTZ on blood glutathione in a blinded, placebo-controlled study of patients with chronic renal failure treated by peritoneal dialysis. Twenty patients were randomly selected to receive OTZ (0.5 g three times a day orally with meals) or placebo for 14 d. Patients visited the clinic for predose blood collection and safety evaluation at baseline (days 3, 7, and 14 and again at 14 d from the last dose [follow-up]). Glutathione concentrations were determined in whole blood by HPLC. OTZ resulted in a significant rise in whole-blood glutathione at days 7 (594 +/- 129 mumol/L) and 14 (620 +/- 108 mumol/L) compared with baseline (544 +/- 139 mumol/L) (P < 0.01 and P < 0.05, respectively). Glutathione was also significantly increased at days 7 and 14 when normalized by hematocrit (Hct) or hemoglobin to correct for anemic status (e.g., 20.7 +/- 5.7 mumol/L per % Hct [day 7] and 20.9 +/- 4.0 mumol/L per % Hct [day 14] versus 18.0 +/- 4.2 mumol/L per % Hct [baseline]; P < 0.05). Glutathione levels did not change in the placebo group at any patient visit, and levels in the OTZ-treated group returned to baseline at follow-up. There were no serious adverse events attributable to OTZ, and the drug appeared to be well tolerated by patients with renal failure treated by continuous ambulatory peritoneal dialysis. Our results show that OTZ increases blood glutathione levels, which may improve antioxidant status in dialysis patients.

Peritoneal dialysis solutions for the 21st century.

Existing peritoneal dialysis (PD) solutions were formulated for the maintenance of fluid and electrolyte balance, correction of metabolic acidosis, and the removal of metabolic waste products. Solutions currently in development are designed to improve biocompatibility by neutralizing pH and reducing glucose degradation products. PD solutions developed for the 21st century will address major clinical needs related to the maintenance of adequate nutrition, improvement of cardiovascular comorbidity, preservation of peritoneal membrane function, and adequacy of dialysis. Nutrineal is the first example of a PD solution to address such a clinical need. Another example is a low sodium solution to improve sodium balance thereby reducing the incidence of hypertension and the need for antihypertensive medication. New solutions under investigation will employ additives to protect the peritoneal membrane during peritonitis and chronic PD and to optimize PD therapy via improvements in ultrafiltration and solute clearances. Examples include the use of antioxidants to reduce oxidative damage during peritonitis and the use of glycosaminoglycans or other additives to enhance ultrafiltration. Future concepts include remodeling of the peritoneum, for example, the use of mesothelial gene therapy to introduce metabolic and anabolic machinery to remove or perpetually recycle metabolic wastes. The 21st century promises to be an exciting and fruitful time for the investigation and development of new products for the improvement of PD therapy.

New developments in peritoneal dialysis solutions.

Existing peritoneal dialysis (PD) solutions were formulated mainly for the maintenance of fluid and electrolyte balance, correction of metabolic acidosis, and the removal of metabolic waste products. New solutions in development, and recently approved in some countries, are designed to improve ultrafiltration during long dwells (polyglucose or icodextrin solutions), to treat malnutrition (amino-acids solutions), and to improve peritoneal biocompatibility (bicarbonate-buffered solutions). Other new solutions under investigation are designed to address unmet clinical needs, including cardiovascular disease and sodium balance, through the use of a low-sodium PD solution; long-term peritoneal viability, through improvements in sterilization processes and the use of nonglucose osmotic agents; and PD adequacy, through the use of solution additives (such as glycosaminoglycans) and tailored PD prescriptions using APD. Future concepts for PD include remodeling of the peritoneum, perhaps using mesothelial gene therapy to introduce metabolic and anabolic machinery to remove or perpetually recycle metabolic wastes.

Maltose and isomaltose in uremic plasma following icodextrin administration.

The presence of mixed disaccharides (maltose and isomaltose) in plasma from uremic patients has been previously investigated using gel-permeation chromatography. However, this method is unable to separate maltose (linked alpha-1-4) from isomaltose (linked alpha-1-6). We describe an alternative method using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) for the direct determination of maltose and isomaltose in uremic plasma. We measured maltose and isomaltose using HPAE-PAD in 6 normal subjects and in 15 uremic patients before and after once-daily icodextrin administration for at least 4 weeks. Both maltose and isomaltose were below limits of detection (< 1.0 mg/L) in plasma from normal controls. Patients with end-stage renal disease treated by continuous ambulatory peritoneal dialysis had elevated levels of isomaltose (23.6 +/- 8.3 mg/L) but low levels of maltose (< 3.0 mg/L). Treatment with icodextrin resulted in elevated plasma levels of maltose (range: 500-1600 mg/L), while levels of isomaltose declined to 9.8 +/- 5.2 mg/L (P < 0.0001 vs. baseline levels). We conclude that isomaltose (not maltose) is the primary disaccharide isomer that is elevated in the plasma of uremic patients, whereas maltose is the primary disaccharide isomer that is elevated following icodextrin administration. Furthermore, icodextrin administration results in an apparent reduction of isomaltose. Additional investigation will be required to address the mechanism for the reduction of isomaltose in patients treated by icodextrin.

Low whole blood and erythrocyte levels of glutathione in hemodialysis and peritoneal dialysis patients.

Dialysis patients are reported to have impaired antioxidant mechanisms, including those involving glutathione-dependent enzymes. This study used high-performance liquid chromatography assays that directly measure total (oxidized + reduced) glutathione and its precursor cysteine (CYS) to compare the whole blood of hemodialysis (prehemodialysis and posthemodialysis) and peritoneal dialysis patients to that of blood donors with no known kidney disease (n=20 in each group). The levels in erythrocytes were calculated from that data (as nmol/g hemoglobin) because these cells are the major compartment of blood glutathione and their survival may be shortened by oxidant damage. Both dialysis groups had significantly (P=0.0001) higher CYS levels in the plasma compartment than the controls (251 nmol/mL), with prehemodialysis levels (432 nmol/mL) being greater than peritoneal dialysis levels (334 nmol/mL). Hemodialysis acutely lowered CYS levels (215 nmol/mL) below those of controls. Expressed per milliliter whole blood, both dialysis groups had significantly (P=0.0001) lower glutathione levels than controls (1,276 nmol/mL), with prehemodialysis and peritoneal dialysis levels being similar (778 and 912 nmol/mL). Values increased prehemodialysis to posthemodialysis, consistent with hemoconcentration. Expressed per gram hemoglobin, the dialysis groups had significantly (P < 0.015) lower glutathione levels than the controls (8,938 nmol/g hemoglobin), with similar prehemodialysis, posthemodialysis, and peritoneal dialysis values (7,207, 7,315, and 7,915 nmol/g hemoglobin, respectively). In summary, hemodialysis and peritoneal dialysis patients are at increased risk from oxidative stress due to glutathione deficiency in whole blood and erythrocytes.

Inhibition of apolipoprotein B and triglyceride secretion in human hepatoma cells (HepG2).

Apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, is assembled into a lipoprotein particle via a complex, multistep process. Recent studies indicate that triglyceride-rich lipoprotein assembly requires the activity of the heterodimeric protein, microsomal triglyceride transfer protein (MTP). We identified a novel inhibitor of apolipoprotein B secretion using the human hepatoma cell line, HepG2. CP-10447, a derivative of the hypnotic drug methaqualone (Quaalude), inhibited apoB secretion from HepG2 cells with an IC50 of approximately 5 microM. CP-10447 also inhibited apoB secretion from Caco-2 cells, a model of intestinal lipoprotein production. In experiments using [3H]glycerol as a precursor for triglyceride synthesis, CP-10447 (20 microM) inhibited radiolabeled triglyceride secretion by approximately 83% (P < 0.0001) in HepG2 cells and 76% (P < 0.05) in Caco-2 cells with no effect on radiolabel incorporation into cellular triglyceride, indicating that CP-10447 inhibited triglyceride secretion without affecting triglyceride synthesis. RNA solution hybridization assay indicated that CP-10447 did not affect apoB or apoA-I mRNA levels. Pulse-chase experiments in HepG2 cells confirmed that CP-10447 inhibited the secretion of apoB (not its synthesis) without affecting secretion of total proteins or albumin and suggested that CP-10447 stimulates the early intracellular degradation of apoB in the endoplasmic reticulum (ER). Further studies demonstrated that CP-10447 is a potent inhibitor of human liver microsomal triglyceride transfer activity (IC50 approximately 1.7 microM) in an in vitro assay containing artificial liposomes and partially purified human MTP. These data suggest that CP-10447 may inhibit apoB and triglyceride secretion by inhibiting MTP activity and stimulating the early ER degradation of apoB. CP-10447 should provide a useful tool for further study of the mechanisms of apoB secretion and triglyceride-rich lipoprotein assembly.

9-cis-retinoic acid increases apolipoprotein AI secretion and mRNA expression in HepG2 cells.

HepG2 cells were studied as a model for regulation of hepatic apolipoprotein AI (apo AI) secretion and gene expression by 9-cis-retinoic acid. HepG2 cells cultured on plastic dishes were exposed to 9-cis-retinoic acid (9-cis-RA) for 48 h with a complete media change at 24 h. Apo AI mass in cultured media was determined by ELISA, by quantitative immunoblotting and by steady-state 35S-methionine labeling. Messenger RNA levels were determined by RNase protection using probes for apo AI and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). 9-cis-RA increased secretion of apo AI by 52% at doses of 10 and 1 microM (6.3 +/- 0.6 vs. 4.2 +/- 0.3; P < 0.005; 6.1 +/- 0.3 vs. 4.0 +/- 0.7 ng of apo AI/mg cell protein, P < 0.05) and by 35% at 0.1 microM (5.5 +/- 0.6 vs. 4.1 +/- 0.4 ng apo AI/mg protein, P < 0.05, n = 4). Immunoblotting results were consistent with results from ELISA (70% increase at 10 microM 9-cis-RA, P < 0.001; 34% increase at 1 microM, P < 0.005, n = 3). Metabolically labeled apoAI in the medium was increased by 39% following steady-state labeling in the presence of 10 microM 9-cis-RA (597 +/- 7 vs. 430 +/- 13 DPM/microliters media; P < 0.001; n = 4). 9-cis-RA (10 microM) also increased HepG2 cell apo AI mRNA expression by 76% (68 700 +/- 400 vs. 38 900 +/- 2700 DPM, P < 0.01, n = 4), whereas expression of G3PDH mRNA was slightly decreased (14%, P < 0.05). Thus, 9-cis-RA stimulates apo AI expression in HepG2 cells, suggesting a role for retinoids in activating endogenous apo AI gene expression.

Low level quantification of cholesteryl ester transfer protein in plasma subfractions and cell culture media by monoclonal antibody-based immunoassay.

Sensitive immunoradiometric (IRMA) and ELISA assays for cholesteryl ester transfer protein (CETP) have been developed using two different monoclonal antibodies (MAbs). The MAbs were prepared against human plasma CETP and demonstrated specificity by their inhibition of cholesteryl ester transfer activity and by immunoblots of crude plasma fractions and whole media from transfected CHO cells. For these sandwich-type assays, one MAb, 2F8, is used for capture, and the second MAb, 2E7, is iodinated (IRMA) or conjugated with alkaline phosphatase (ELISA) and used for detection. Both assays are linear and provide sensitivities much greater than previously reported. The IRMA allows for the accurate quantification of CETP in the range of 0.5-20 ng/assay (5-200 ng/ml), the ELISA 0.05-5 ng/assay (0.5-50 ng/ml). Using the IRMA, the mean plasma CETP concentration in 44 normolipidemic individuals was determined to be 2.10 +/- 0.36 micrograms/ml; 2.05 +/- 0.33 for males (n = 25) and 2.16 +/- 0.40 for females (n = 19). Values ranged from 1.28 to 2.97 micrograms/ml and CETP mass correlated well with cholesteryl ester transfer activity (r = 0.913, n = 23). The distribution of CETP in human plasma was examined both by gel permeation fast protein liquid chromatography (FPLC) and by native gel electrophoresis. For FPLC using agarose resins, a low ionic strength, isotonic buffer system resulted in near total recoveries of CETP, and demonstrated a peak for CETP mass centered at molecular masses of 150 to 180 kilodaltons, larger than that for free monomeric CETP. Native acrylamide gel electrophoresis of plasma from six individuals, followed by 2F8/2E7 sandwich immunoblotting, showed CETP migrating within a size range of 170-220 kilodaltons. This result is consistent with suggestions that plasma CETP is associated with small-sized HDL. Agarose gel electrophoresis showed plasma CETP, as well as purified recombinant CETP, to be prebeta migrating. For determining the concentration of CETP in the media of cultured HepG2 cells, advantage was taken of the high sensitivity of the ELISA. CETP levels were found to increase linearly over the 100-h culture period, reaching 8.0 +/- 0.4 ng/ml (18.0 +/- 1.3 ng/mg cell protein). These sensitive, direct immunoassays for CETP mass should be valuable aids for examining the behavior of CETP in plasma and other complex systems, as well as for studying the synthesis and secretion of CETP by different cells and tissues.

Inhibition of cholesteryl ester transfer protein activity in hamsters alters HDL lipid composition.

We investigated the role of cholesteryl ester transfer protein (CETP) in hamsters by using a monoclonal antibody (MAb) that inhibited hamster CETP activity. MAbs were prepared against partially purified human CETP and screened for inhibiton of 3H-cholesteryl oleate (CE) transfer from LDL to HDL in the presence of human plasma bottom fraction (d > 1.21 g/ml). Antibody 1C4 inhibited CE transfer activity in both human plasma bottom fraction (IC50 = approximately 4 micrograms/ml) and in whole plasma from male Golden Syrian hamsters (IC50 = approximately 30 micrograms/ml). Purified MAb 1C4 was injected into chow- and cholesterol-fed hamsters, and blood was collected for analysis of plasma CETP activity and HDL lipid composition. Plasma CETP activity was inhibited by 70%-80% at all and HDL lipid composition. Plasma CETP activity was inhibited by 70%-80% at all times up to 24 h following injection of 500 micrograms MAb 1C4 (approximately 3.7 mg/kg). The amount of antibody required for 50% inhibition at 24 h post-injection was 200 micrograms (approximately 1.5 mg/kg). Inhibition of hamster CETP activity in vivo increased hamster HDL cholesterol by 33% (P < 0.0001), increased HDL-CE by 31% (P < 0.0001) and decreased HDL-triglyceride by 42% (P < 0.0001) (n = 36) as determined following isolation of HDL by ultracentrifugation. An increase in HDL cholesterol and a redistribution of cholesterol to a larger HDL particle were also observed following fast protein liquid chromatography (FPLC) gel filtration of plasma lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Transepithelial transport of cholyltaurine by Caco-2 cell monolayers is sodium dependent.

Bile acids are efficiently recovered from the intestinal lumen by a Na(+)-dependent transport process that is localized in the ileal enterocyte brush-border membrane. To establish a cell culture model for this process, we examined the Na+ dependence of cholyltaurine (C-tau; taurocholate) transport across monolayers of differentiated Caco-2 cells grown on permeable filter inserts. Transport of [3H]C-tau was Na+ dependent (> 20-fold stimulation), saturable, and time linear for at least 60 min. The apparent Michaelis constant of [3H]C-tau transport was approximately 65 microM, and the maximal transport rate was approximately 800 pmol.min-1.mg protein-1. Transport of [3H]C-tau in the apical-to-basolateral direction was 17-fold greater than transport in the reverse direction. Lowered incubation temperature, various metabolic inhibitors, and various unlabeled bile acids inhibited [3H]C-tau transport. Caco-2 cells thus transport bile acids in a manner similar to that described for ileal brush-border membrane vesicles and isolated ileal enterocytes and are therefore an appropriate model for studying the molecular basis of ileal bile acid transport.

Apolipoprotein expression and cellular differentiation in Caco-2 intestinal cells.

Caco-2 cells, cultured for 18 days on porous filter supports and conventional plastic culture dishes, were used to study the effects of cellular differentiation on the expression of apolipoprotein (apo) genes. Media of filter-grown cells accumulated more apo B as apo B-48 and contained three times the amount of edited apo B mRNA compared with plastic-grown cells. The accumulation of apo A-I by media of plastic-grown cells was higher than accumulation by filter-grown cells, despite similar concentrations of apo A-I mRNA. The apo A-IV was detectable in the culture media earlier with filter-grown cells compared with plastic-grown cells, despite similar apo A-IV mRNA concentrations. Plastic-grown cells contained more apo E mRNA, and their media accumulated more apo E than filter-grown cells. With the exception of apo A-I, apo gene expression changed with Caco-2 cell differentiation to resemble more closely the patterns seen in adult enterocytes. There were no effects or minimal effects of added retinoic acid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or thyroid hormone on apo accumulation in media of filter-grown cultures of Caco-2 cells. However, 1,25(OH)2D3 and thyroid hormone increased apo B, apo A-IV, and apo A-I mRNA concentrations, retinoic acid increased apo B mRNA concentrations alone, and all three reduced apo E mRNA concentrations. Ratios of edited to unedited apo B mRNA were unaffected. In conclusion, culture substratum importantly influences Caco-2 cell differentiation. Soluble factors that influence cellular differentiation may affect apo gene expression over and above effects mediated by the culture substratum.(ABSTRACT TRUNCATED AT 250 WORDS)

Editing of apolipoprotein B messenger RNA in differentiated Caco-2 cells.

During the differentiation of human enterocytes, the secretion of apolipoprotein B is switched from the apoB-100 form seen almost exclusively in fetal cells to the apoB-48 form seen almost exclusively in adult cells. This switch is accomplished by the post-transcriptional editing of the messenger RNA for apoB-100. We report that a similar switch occurs during the differentiation of the human colonic carcinoma cell line, Caco-2, and that this is accomplished by the same mRNA editing mechanism. Caco-2 cells cultured on Millipore filters developed confluent electrically resistant monolayers, and on Western blot analysis, using a monoclonal antibody directed against the amino terminal region of human apoB-100 (Mab C1.4), secreted greater than 50% apoB-48 (of total apoB-100) into culture media, while Caco-2 cells grown on plastic secreted greater than 95% apoB-100. To assess whether mRNA editing was responsible for the switchover from apoB-100 to apoB-48, apoB cDNA fragments spanning nucleotides 6504-6784 of apoB mRNA were synthesized using RNA isolated from Caco-2 cells grown on filters and Caco-2 cells grown on plastic. The appropriate oligonucleotide primers and Moloney murine leukemia virus reverse transcriptase were used. The resulting cDNA fragments were amplified by the polymerase chain reaction (PCR), and PCR products were subcloned and sequenced. A single cytosine/thymine base change occurred in 8/20 clones of cDNA derived from Caco-2 cells grown on filters (corresponding to a cytosine/uridine change in mRNA) and in 1/25 clones of cDNA derived from Caco-2 cells grown on plastic. PCR products of genomic sequences from Caco-2 cells did not contain the stop codon.(ABSTRACT TRUNCATED AT 250 WORDS)

Oleic acid stimulation of apolipoprotein B secretion from HepG2 and Caco-2 cells occurs post-transcriptionally.

HepG2 and Caco-2 cells were studied to compare the regulation of liver and intestinal apolipoprotein (apo) biosynthesis and secretion by fatty acids. Incubation with fatty acid consistently stimulated apoB production by both HepG2 and Caco-2 cells. Media concentrations of apoB, determined by radioimmunoassay, were approx. 3-fold greater for cells incubated for 24 h in serum-free medium containing oleic acid bound to albumin than for cells incubated with albumin alone. Oleic acid also resulted in a 2-3-fold accumulation of cellular triacylglycerol for HepG2 cells and Caco-2 cells. Cellular apoB and media and cellular apoA-I concentrations were not affected by oleic acid. Immunoblotting with a monoclonal antibody against human apoB confirmed a greater mass of apoB in media from HepG2 and Caco-2 cells incubated with oleic acid. Radiolabeled apoB-100 was also increased in media from HepG2 and Caco-2 cells incubated with [35S]methionine for 24 h in the presence of oleic acid, suggesting enhanced apoB synthesis. However, apoB mRNA concentrations were unchanged in response to oleic acid. Gel filtration of media by fast protein liquid chromatography (FPLC) revealed a redistribution of apoB from LDL-sized particles to VLDL or chylomicrons in media from Caco-2 cells incubated with oleic acid, whereas apoB remained in LDL for HepG2 cells. ApoA-I in media from HepG2 and Caco-2 cells eluted as free or lipid-poor apoA-I, and the apoA-I distribution was unaltered by incubation with oleic acid. These data demonstrate that HepG2 and Caco-2 cells maintained in supplemented serum-free medium respond to oleic acid by a similar post-transcriptional increase in apoB synthesis, but different packaging of apoB into triacylglycerol-rich lipoproteins.