Pediatric reference intervals for endocrine markers in healthy children and adolescents on the Liaison XL (DiaSorin) immunoassay system.

OBJECTIVES: Prominent physiological changes occurring throughout childhood and adolescence necessitate the consideration of age and sex in biomarker interpretation. Critical gaps exist in pediatric reference intervals (RIs) for specialized endocrine markers, despite expected influence of growth and development. The current study aimed to establish and/or verify RIs for six specialized endocrine markers on a specialized immunoassay system. METHODS: Samples were collected from healthy children and adolescents (5 to <19 years) and apparently healthy outpatients (0 to <5 years) as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER). Serum samples were analysed for aldosterone, renin (plasma), thyroglobulin, anti-thyroglobulin, growth hormone, and insulin-like growth factor-1 (IGF-1) on the Liaison XL (DiaSorin) immunoassay platform. RIs (2.5th and 97.5th percentiles) were established for aldosterone, renin, thyroglobulin, anti-thyroglobulin, and growth hormone. Manufacturer-recommended pediatric RIs for IGF-1 were verified. RESULTS: Age-specific RIs were established for aldosterone, renin, and thyroglobulin, while no age-specific differences were observed for anti-thyroglobulin or growth hormone. IGF-1 was the only endocrine marker studied that demonstrated significant sex-specific differences. Manufacturer-recommended IGF-1 RIs were verified for children aged 6 to <19 years, while those for children aged 0 to <6 years did not verify. CONCLUSIONS: This study marks the first time that pediatric RIs for aldosterone and renin were established in the CALIPER cohort and highlights the dynamic changes that occur in water and sodium homeostasis during the first years of life. Overall, these data will assist pediatric clinical laboratories in test result interpretation and improve clinical decision-making for patients tested using Liaison immunoassays.

Pediatric reference intervals for calculated LDL cholesterol, non-HDL cholesterol, and remnant cholesterol in the healthy CALIPER cohort.

OBJECTIVES: Increased prevalence of pediatric obesity and associated co-morbidities has heightened the concern for cardiovascular disease (CVD) risk later in life. Although the fasting lipid profile is traditionally used to assess CVD risk, the non-fasting lipid profile may simplify lipid testing and better predict CVD risk. Unfortunately, non-fasting lipid reference values are limited, particularly for children. The Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) has recruited thousands of healthy pediatric subjects to develop a pediatric reference interval database. Here, CALIPER reports pediatric reference intervals for non-fasting calculated low-density lipoprotein cholesterol (LDLc), non-high-density lipoprotein cholesterol (non-HDLc) and remnant cholesterol. METHODS: Non-fasting serum samples from the CALIPER cohort of community children and adolescents were previously analyzed for HDLc, total cholesterol, and triglycerides. These values were used to calculate LDLc, non-HDLc, and remnant cholesterol and subsequently establish reference intervals with corresponding 90% confidence intervals according to CLSI EP28-A3c guidelines. Reference intervals were also calculated using alternative statistical methods highlighted in recent literature. RESULTS: All three lipid parameters required an age partition at 1 year due to wider reference intervals in the first year of life. LDLc and non-HDLc required sex partitioning for subjects 1-<10 years. Non-HDLc upper reference limit was higher than the 2011 National Heart, Lung, and Blood Institute (NHLBII) pediatric recommended cut-offs, suggesting elevated atherogenic lipoproteins in a proportion of apparently healthy pediatric subjects. The LDLc upper reference limit (10-<19 year partition) was the same as the NHLBI cut-off, potentially due to lower calculated LDLc values in the non-fasting state. CONCLUSIONS: With the increased use of non-fasting lipid profiles, age- and sex-specific reference intervals and appropriate clinical decision limits are necessary for pediatric lipid monitoring. Our data supports the notion that appropriate decision limits, rather than reference intervals, should be used to interpret lipid levels in children as there is a high prevalence of hyperlipidemia in the apparently healthy pediatric population.

A sensitive and rapid mass spectrometric method for the simultaneous measurement of eight steroid hormones and CALIPER pediatric reference intervals.

OBJECTIVES: To develop an accurate assay and establish the normal reference intervals for serum cortisol, corticosterone, 11-deoxycortisol, androstenedione, 21-hydroxyprogesterone, testosterone, 17-hydroxyprogesterone, and progesterone. These steroids are commonly used as biomarkers for the diagnosis and monitoring of endocrine diseases such as congenital adrenal hyperplasia. Appropriate age- and gender-stratified reference intervals are essential in accurate interpretation of steroid hormone levels. DESIGN AND METHODS: The samples analyzed in this study were collected from healthy, ethnically diverse children in the Greater Toronto Area as part of the CALIPER program. A total of 337 serum samples from children between the ages of 0 and 18years were analyzed. The concentrations were measured by using an LC-MS/MS method. The data were analyzed for outliers and age- and gender-specific partitions were established prior to establishing the 2.5th and 97.5th percentiles for the reference intervals. RESULTS: Reference intervals for all hormones required significant age-dependent stratification while testosterone and progesterone required additional sex-dependent stratification. CONCLUSIONS: We report a sensitive, accurate and relatively fast LC-MS/MS method for the simultaneous measurement of eight steroid hormones. Detailed reference intervals partitioned based on both age and gender were also established for all eight steroid hormones.

Influence of fasting and sample collection time on 38 biochemical markers in healthy children: a CALIPER substudy.

OBJECTIVES: Fasting samples can be difficult to obtain in the pediatric setting, particularly in neonates. As part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER), we aimed to determine if there are differences in serum concentrations of pediatric biochemical markers measured at fasting, postprandial, and random time points throughout the day. DESIGN AND METHODS: Blood was drawn from 27 healthy children and adolescents (aged 4-18) with informed consent at 4 time points: after overnight fast, mid-morning after breakfast, within 2h after lunch, and late afternoon. The effect of fasting on 38 chemistries was evaluated by paired, two-tailed student'st-tests. Analysis of the effect of time of day was done using paired, repeated-measures ANOVA. RESULTS: Fasting significantly affected 22 analytes, with HDL cholesterol being the most highly affected. Values tended to decrease postprandially, except for five analytes, including triglycerides, which increased. By ANOVA, 28 chemistries significantly differed across times of day tested. CONCLUSIONS: Fasting is necessary for analysis of certain chemistries in pediatric subjects. Pediatricians should consider diurnal factors when ordering non-fasting tests and interpreting test results.

Early determinants of atherosclerosis in paediatric systemic lupus erythematosus.

OBJECTIVES: To assess traditional and non-traditional cardiovascular risk factors and to determine the prevalence and correlates of early vascular markers of atherosclerosis in paediatric systemic lupus erythematosus (pSLE). METHODS: Fifty-four adolescents with pSLE had cardiovascular risk factor assessment, disease activity and vascular testing including carotid intima-media thickness (CIMT), flow-mediated dilatation (FMD), arterial stiffness measures, and myocardial perfusion studies. RESULTS: The traditional risk factors of hypertension, elevated triglycerides, apolipoprotein B, haemoglobin A1c and insulin levels and non-traditional risk factors of elevated homocysteine and fibrinogen were present (all p<0.001). Some arterial stiffness measures, central pulse wave velocity and characteristic impedance were elevated (p<0.001), but CIMT, FMD and myocardial perfusion were normal. Cumulative prednisone dose correlated with total cholesterol (r=0.5790, p<0.001) and elevated LDL-C (r=0.4488, p=0.0012). Hydroxychloroquine treatment correlated negatively with total cholesterol (r=-0.4867, p=0.0002), LDL-C (r=-0.4805, p=0.0002) and apolipoprotein B (r=-0.4443, p=0.0011). In multivariate analysis LDL-C correlated with cumulative prednisone dose and negatively with hydroxychloroquine treatment (R2=0.40, p<0.001). CONCLUSIONS: An increased burden of traditional and non-traditional risk factors and early evidence of insulin resistance and increased central arterial stiffness were present in paediatric SLE. Disease-specific and therapy-related factors are likely modifying these cardiovascular risk profiles warranting prospective longitudinal studies.

Continuous bupivacaine infusion post-iliac crest bone graft harvesting in pediatric cleft surgery: role and comparison with ketorolac.

OBJECTIVE: To investigate the use of intravenous ketorolac and iliac crest bupivacaine infusion in the management of iliac crest donor-site pain in the pediatric cleft population. The null hypothesis was there is no difference with respect to pain scores between ketorolac and iliac crest bupivacaine infusion as analgesic adjuncts to intravenous opioids. METHODS: A total of 54 children and adolescents (27 boys, 27 girls) undergoing alveolar cleft repair or Le Fort I osteotomy were assigned randomly in a prospective, single-blinded fashion to one of three groups: intravenous ketorolac plus iliac crest normal saline infusion, intravenous ketorolac plus iliac crest bupivacaine infusion, or iliac crest bupivacaine infusion alone. Iliac crest infusions and ketorolac were administered for 48 hours or until discharge, whichever occurred first. All patients received morphine via a patient-controlled analgesia device. MAIN OUTCOME MEASURE(S): Primary outcome was pain score, and secondary outcomes were morphine consumption and satisfaction scores. RESULTS: Pain scores, morphine consumption, and satisfaction scores were not significantly different among groups. Estimated costs were significantly higher for bupivacaine infusion than intravenous ketorolac. CONCLUSIONS: Iliac crest donor-site pain is well managed in this patient population. Intravenous ketorolac and iliac crest bupivacaine infusion provide comparable analgesia for iliac crest bone graft donor-site pain in children and adolescents.

An estimating on of the economical value of Arsanjan Bonab Forest Park, Iran.

Using of amusement places always has been important for humankind. Therefore, assessment of value for this promenaded and usage of nature is necessary for future programming in management of natural resources. For this reason, this research will discuss an assessment of the promenaded value of Bonab Forest Park and determines a visitor's Willingness To Pay (WTP) for promenaded benefits obtained. In this study, two techniques are used for Contingent Valuation (CV) $ Travel Cost (TC) method by Dichotomous Choice (DC). For determination of visitor's willingness to pay simple average method was employed. Results indicate that 94.3% of visitors are willing to pay for promenaded values at the Bonab Forest Park. The mean value for willingness to pay for the value of the park is 0.38 $ for each person per month. Annual value for promenade in this park was calculated 3439.3 $ per hector. Other important results in this research show that forest park has promenaded value significantly and on the other hand, we can increase the usage, protection and numbers of tourists.

The glucagon-like peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors attenuate postprandial lipaemia through mechanisms that remain unclear. As dyslipidaemia is a contributing risk factor for cardiovascular disease in type 2 diabetes, we examined the mechanisms linking pharmacological and physiological regulation of GLP-1 action to control of postprandial lipid metabolism. METHODS: Postprandial lipid synthesis and secretion were assessed in normal and fructose-fed hamsters and in wild-type mice that were treated with or without sitagliptin. Apolipoprotein B-48 (ApoB-48) synthesis and secretion were also examined in primary enterocyte cultures. The importance of exogenous vs endogenous GLP-1R signalling for regulation of intestinal lipoprotein synthesis and secretion was assessed in mice and hamsters treated with the GLP-1R agonist exendin-4, the GLP-1R antagonist exendin(9-39) and in Glp1r (+/+) vs Glp1r (-/-) mice. RESULTS: Sitagliptin decreased fasting plasma triacylglycerol, predominantly in the VLDL fraction, as well as postprandial triacylglycerol-rich lipoprotein (TRL)-triacylglycerol, TRL-cholesterol and TRL-ApoB-48 in hamsters and mice. GLP-1R activation with exendin-4 alone also decreased plasma and TRL-ApoB-48 in hamsters and mice, and reduced secretion of ApoB-48 in hamster enterocyte cultures. Conversely, blockade of endogenous GLP-1R signalling by the antagonist exendin(9-39) or genetic elimination of GLP-1R signalling in Glp1r (-/-) mice enhanced TRL-ApoB-48 secretion in vivo. Co-administration of exendin(9-39) also abolished the hypolipidaemic effect of sitagliptin. CONCLUSIONS/INTERPRETATION: Potentiation of endogenous incretin action via DPP-4 inhibition or pharmacological augmentation of GLP-1R signalling reduces intestinal secretion of triacylglycerol, cholesterol and ApoB-48. Moreover, endogenous GLP-1R signalling is essential for the control of intestinal lipoprotein biosynthesis and secretion.

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: evidence for PKC activation and oxidative stress-activated signaling pathways.

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine(307) phosphorylation of IRS-1. IRS-1 Serine(307) phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IkappaB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKalpha/beta, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.

Does the Macroduct collection system reliably define sweat chloride concentration in subjects with intermediate results?

OBJECTIVES: The sweat test remains the current diagnostic gold standard for CF disease. Many CF testing centres have switched from the Gibson and Cooke to the Macroduct. Since the validity and sensitivity of Macroduct has not been tested in patients with intermediate sweat chloride concentrations, we compared both methods simultaneously including subjects expected to have intermediate results. DESIGN AND METHODS: We prospectively evaluated controls, obligate heterozygotes, patients with CF and with an uncertain diagnosis of CF (congenital absence of the vas deferens, pancreatitis and sinopulmonary disease). RESULTS: We assessed 82 subjects (3.7-60.1 years); 14 healthy controls, 7 obligate heterozygotes, 20 CF (15 pancreatic insufficient, 5 pancreatic sufficient), and 41 with unproven diagnosis. Mean test difference was close to 0 (95% CI+/-20 mmol/L) and test values were highly correlated (r=0.93, p < or =0.0001). Discrepancies between the two testing methods occurred in 22% of subjects. CONCLUSION: Sweat chloride measured by Macroduct highly correlates with Gibson and Cooke for concentrations in all ranges, including the intermediate range. This study reveals the limitations of sweat testing for excluding a diagnosis of CF since 38% of subjects had intermediate range results.

Mechanisms of hepatic very low-density lipoprotein overproduction in insulin resistance.

An important complication of insulin-resistant states, such as obesity and type 2 diabetes, is an atherogenic dyslipidemia profile characterized by hypertriglyceridemia, low plasma high-density lipoproteins (HDL) cholesterol and a small, dense low-density lipoprotein (LDL) particle profile. The physiological basis of this metabolic dyslipidemia appears to be hepatic overproduction of apoB-containing very low-density lipoprotein (VLDL) particles. This has focused attention on the mechanisms that regulate VLDL secretion in insulin-resistant states. Recent studies in animal models of insulin resistance, particularly the fructose-fed hamster, have enhanced our understanding of these mechanisms, and certain key factors have recently been identified that play important roles in hepatic insulin resistance and dysregulation of the VLDL secretory process. This review focuses on these recent developments as well as on the hypothesis that an interaction between enhanced flux of free fatty acids from peripheral tissues to liver, chronic up-regulation of de novo lipogenesis by hyperinsulinemia and attenuated insulin signaling in the liver may be critical to the VLDL overproduction state observed in insulin resistance. It should be noted that the focus of this review is on molecular mechanisms of the hypertriglyceridemic state associated with insulin resistance and not that observed in association with insulin deficiency (e.g., in streptozotocin-treated animals), which appears to have a different etiology and is related to a catabolic defect rather than secretory overproduction of triglyceride-rich lipoproteins.

Triglyceride enrichment of HDL does not alter HDL-selective cholesteryl ester clearance in rabbits.

Triglyceride (TG) enrichment of high density lipoprotein (HDL), which occurs in hypertriglyceridemic states, significantly enhances the rate at which HDL apolipoprotein (apo)A-I is cleared from the circulation of healthy humans. In the New Zealand White (NZW) rabbit, a species naturally deficient in hepatic lipase (HL), TG enrichment of HDL requires prior lipolytic modification to enhance apoA-I clearance. However, the effect of TG enrichment of HDL on the subsequent clearance of HDL cholesteryl ester (CE) has not previously been examined in vivo. Therefore, we investigated, in the NZW rabbit, the effect of ex vivo TG enrichment of rabbit HDL (by incubation with human very low density lipoprotein) on the clearance of HDL CE and apoA-I radiolabeled with (3)H-cholesteryl oleyl ether and with (131)I, respectively. In nine experiments, TG enrichment of rabbit HDL resulted in an 87% average increase in HDL TG and a corresponding 31% reduction in HDL CE content. The calculated apoA-I and CE fractional catabolic rates associated with TG-rich versus fasting HDL tracers were not significantly different (apoA-I: 0.119 +/- 0.017 vs. 0.107 +/- 0.024 pools per h, P = 0.68; CE: 0.147 +/- 0.014 vs. 0.114 +/- 0.019 pools per h, P = 0.20). In an animal model deficient in HL, TG enrichment of HDL did not alter the rates of HDL apoA-I or selective CE clearance. Further studies are needed to determine whether, in the presence of HL, TG enrichment of HDL alters selective HDL CE clearance.

Modulation of hepatic lipoprotein synthesis and secretion by taxifolin, a plant flavonoid.

In the present study, the effects of taxifolin, a plant flavonoid, on lipid, apolipoprotein B (apoB), and apolipoprotein A-I (apoA-I) synthesis and secretion were determined in HepG2 cells. Pretreatment of cells with (+/-)-taxifolin led to an inhibition of cholesterol synthesis in a dose- and time-dependent manner, with an 86 +/- 3% inhibition at 200 microM observed within 24 h. As to the mechanism underlying this inhibitory effect, taxifolin was shown to inhibit the activity of HMG-CoA reductase by 47 +/- 7%. In addition, cellular cholesterol esterification, and triacylglycerol and phospholipid syntheses, were also significantly suppressed in the presence of taxifolin. ApoA-I and apoB synthesis and secretion were then studied by pulse-chase experiments. ApoA-I secretion was found to increase by 36 +/- 10%. In contrast, an average reduction of 61 +/- 8% in labeled apoB in the medium was apparent with taxifolin. This effect on secretion appeared not to be exerted at the transcriptional level. Rather, the effect on apoB secretion was found to be exerted in the early stages of apoB degradation and to be sensitive to dithiothreitol (DTT) and insensitive to N-acetyl-leucyl-leucyl-norleucinal, suggesting a proteolytic pathway involving a DTT-sensitive protease. Fractionation of secreted apoB revealed a slight shift in the distribution of secreted apoB-containing lipoproteins. Cholesteryl ester, rather than triacylglycerol, was shown to be the lipid that primarily regulated apoB secretion. In summary, our data suggest that taxifolin decreases hepatic lipid synthesis with a concomitant decrease and increase in apoB and apoA-I secretion, respectively.

Translocational status of ApoB in the presence of an inhibitor of microsomal triglyceride transfer protein.

Despite numerous studies demonstrating that microsomal triglyceride transfer protein (MTP) activity is critical to apoB secretion, there is still controversy as to whether MTP directly facilitates the translocation of apoB across the membrane of the endoplasmic reticulum (ER) through either the recruitment of lipids and/or chaperone activity. In the present study, a specific inhibitor of MTP (BMS 197636) was utilized in HepG2 cells to investigate whether a direct relationship exists between the translocation of apoB across the ER membrane and the lipid-transferring activity of MTP. Inhibition of MTP (with 10 and 50 nmol/L of the inhibitor) did not significantly affect the translocation of newly synthesized apoB (P = 0.77) or the translocational efficiency of the steady-state apoB mass (P = 0.45), despite a 49% decrease in apoB secretion and increased proteosomal degradation. These results compared well with subcellular fractionation experiments which showed no significant change in the fraction of apoB accumulated in the lumen of isolated microsomes in MTP-treated cells (P = 0.35). In summary, MTP lipid transfer activity does not appear to influence translocational status of apoB, but its inhibition is associated with an increased susceptibility to proteasome-mediated degradation and reduced assembly and secretion of apoB lipoprotein particles.

Intracellular mechanisms regulating apoB-containing lipoprotein assembly and secretion in primary hamster hepatocytes.

We studied the biogenesis of apolipoprotein B (apoB) in primary hepatocytes isolated from hamster liver, an animal model with striking resemblance to humans in lipoprotein metabolism. Hamster hepatocytes were found to assemble and secrete apoB-containing lipoproteins at a density of VLDL. Intracellular mechanisms of apoB biogenesis were investigated in both intact and permeabilized hamster hepatocytes. Translocational status of hamster apoB-100 was examined using trypsin protection assays in permeabilized cells as well as isolated microsomes which revealed that 27-42% of newly synthesized apoB was trypsin accessible as opposed to a control protein, transferrin, which was found to be essentially insensitive to exogenous trypsin. Subcellular fractionation of membrane and lumenal apoB pools indicated, however, that only a minor fraction of hamster apoB was associated with the microsomal membrane. Approximately 40% of newly synthesized apoB was found to be degraded post-translationally in a process sensitive to MG132. Immunoblotting analysis of apoB immunoprecipitates revealed ubiquitination of hamster apoB suggesting the involvement of the proteasome in its intracellular turnover. In addition to MG132, o-phenanthroline, a metalloprotease inhibitor, was also effective in stabilizing hamster apoB. Experiments in permeabilized hamster hepatocytes further confirmed post-translational instability of hamster apoB which was degraded over a 3-h chase generating proteolytic fragments including 167, 70, 57, and 46 kDa intermediates. Of these only the 70 kDa fragment was ALLN sensitive. Oleate treatment of hamster hepatocytes provided protection against intracellular apoB degradation, but did not stimulate its extracellular secretion. ApoB was assembled in the microsomal lumen into lipoprotein particles with densities of LDL and VLDL which were subsequently secreted as VLDL with a minor fraction forming HDL-like particles. In summary, hamster hepatocytes appear to efficiently assemble and secrete apoB-containing VLDL, although a significant pool of newly synthesized apoB is retained intracellularly and becomes sensitive to proteasome-mediated degradation as well as other proteases in the secretory pathway, generating specific degradative intermediates.

Mechanisms of hepatic very low density lipoprotein overproduction in insulin resistance. Evidence for enhanced lipoprotein assembly, reduced intracellular ApoB degradation, and increased microsomal triglyceride transfer protein in a fructose-fed hamster model.

A novel animal model of insulin resistance, the fructose-fed Syrian golden hamster, was employed to investigate the mechanisms mediating the overproduction of very low density lipoprotein (VLDL) in the insulin resistant state. Fructose feeding for a 2-week period induced significant hypertriglyceridemia and hyperinsulinemia, and the development of whole body insulin resistance was documented using the euglycemic-hyperinsulinemic clamp technique. In vivo Triton WR-1339 studies showed evidence of VLDL-apoB overproduction in the fructose-fed hamster. Fructose feeding induced a significant increase in cellular synthesis and secretion of total triglyceride (TG) as well as VLDL-TG by primary hamster hepatocytes. Increased TG secretion was accompanied by a 4.6-fold increase in VLDL-apoB secretion. Enhanced stability of nascent apoB in fructose-fed hepatocytes was evident in intact cells as well as in a permeabilized cell system. Analysis of newly formed lipoprotein particles in hepatic microsomes revealed significant differences in the pattern and density of lipoproteins, with hepatocytes derived from fructose-fed hamsters having higher levels of luminal lipoproteins at a density of VLDL versus controls. Immunoblot analysis of the intracellular mass of microsomal triglyceride transfer protein, a key enzyme involved in VLDL assembly, showed a striking 2.1-fold elevation in hepatocytes derived from fructose-fed versus control hamsters. Direct incubation of hamster hepatocytes with various concentrations of fructose failed to show any direct stimulation of its intracellular stability or extracellular secretion, further supporting the notion that the apoB overproduction in the fructose-fed hamster may be related to the fructose-induced insulin resistance in this animal model. In summary, hepatic VLDL-apoB overproduction in fructose-fed hamsters appears to result from increased intracellular stability of nascent apoB and an enhanced expression of MTP, which act to facilitate the assembly and secretion of apoB-containing lipoprotein particles.

Tocotrienol: a review of its therapeutic potential.

OBJECTIVES: To summarize new knowledge surrounding the physiological activity of tocotrienol, a natural analogue of tocopherol. RESULTS: The biological activity of vitamin E has generally been associated with its well-defined antioxidant property, specifically against lipid peroxidation in biological membranes. In the vitamin E group, alpha-tocopherol is considered to be the most active form. However, recent research has suggested tocotrienol to be a better antioxidant. Moreover, tocotrienol has been shown to possess novel hypocholesterolemic effects together with an ability to reduce the atherogenic apolipoprotein B and lipoprotein(a) plasma levels. In addition, tocotrienol has been suggested to have an anti-thrombotic and anti-tumor effect indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer. CONCLUSION: The physiological activities of tocotrienol suggest it to be superior than alpha-tocopherol in many situations. Hence, the role of tocotrienol in the prevention of cardiovascular disease and cancer may have significant clinical implications. Additional studies on its mechanism of action, as well as, long-term intervention studies, are needed to clarify its function. From the pharmacological point-of-view, the current formulation of vitamin E supplements, which is comprised mainly of alpha-tocopherol, may be questionable.

Studies on degradative mechanisms mediating post-translational fragmentation of apolipoprotein B and the generation of the 70-kDa fragment.

It has been well established that the biogenesis of apoB is mediated co-translationally by the cytosolic proteasome. Here, however, we investigated the role of both the cytosolic proteasome as well as non-proteasome-mediated degradation systems in the post-translational degradation of apoB. In pulse-chase labeling experiments, co-translational (0-h chase) apoB degradation in both intact and permeabilized cells was sensitive to proteasome inhibitors. Interestingly, turnover of apoB in intact cells over a 2-h chase was partially inhibitable by lactacystin, thus suggesting a role for the cytosolic proteasome in the post-translational degradation of apoB. In permeabilized cells, however, there was no post-translational protection of apoB by lactacystin. Further investigations of proteasomal activity in HepG2 cells revealed that, following permeabilization, there was a dramatic loss of the 20 S proteasomal subunits, and consequently the cells exhibited no detectable lactacystin-inhibitable activity. Thus, apoB fragmentation and the generation of the 70-kDa apoB degradation fragment, characteristic of permeabilized cells, continued to occur in these cells despite the absence of functional cytosolic proteasome. Similar results were observed when we used a derivative of lactacystin, clastolactacystin beta-lactone, which represents the active species of the inhibitor. Interestingly, however, the abundance of the 70-kDa fragment could be modulated by the microsomal triglyceride transfer protein inhibitor, BMS-197636, as well as by pretreatment of the permeabilized cells with dithiothreitol. These data thus suggest that although the cytosolic proteasome appears to be involved in the post-translational turnover of apoB in intact cells, the specific post-translational fragmentation of apoB generating the 70-kDa fragment observed in permeabilized cells occurs independent of the cytosolic proteasome.

Effects of gamma-tocotrienol on ApoB synthesis, degradation, and secretion in HepG2 cells.

gamma-Tocotrienol (gamma-T3), a naturally occurring analog of tocopherol (vitamin E), has been shown to have a hypocholesterolemic effect in animals and humans. Unlike tocopherol, it has also been shown to reduce plasma apoB levels in hypercholesterolemic subjects. The aim of this study was to define the mechanism of action of gamma-T3 on hepatic modulation of apoB production using cultured HepG2 cells as the model system. HepG2 cells preincubated with gamma-T3 were initially shown to inhibit the rate of incorporation of [14C]acetate into cholesterol in a concentration- and time-dependent manner, with a maximum 86+/-3% inhibition at 50 micromol/L observed within 6 hours. gamma-T3, on the other hand, had no significant effect on the uptake of [14C]glycerol into pools of cellular triacylglycerol and phospholipid relative to untreated control. The rate of apoB synthesis and secretion was then studied by an [35S]methionine pulse-labeling experiment and quantified by immunoprecipitating apoB on chasing up to 3 hours. An average reduction of 24+/-3% in labeled apoB in the media was apparent with gamma-T3 despite a 60+/-2% increase in apoB synthesis. Fractionation of secreted apoB revealed a relatively denser lipoprotein particle, suggesting a less stable particle. Using a digitonin-permeabilized HepG2 cell system, the effects of gamma-T3 on apoB translocation and degradation in the endoplasmic reticulum were further investigated. The generation of a specific N-terminal 70-kDa proteolytic fragment proved to be a sensitive measure of the rate of apoB translocation and degradation. The abundance of this fragment increased significantly in gamma-T3-treated cells relative to untreated control cells (50+/-21%) after 2 hours of chase. In addition, the presence of gamma-T3 resulted in an average decrease of 64+/-8% in intact apoB. Taken together, the data suggest that gamma-T3 stimulates apoB degradation possibly as the result of decreased apoB translocation into the endoplasmic reticulum lumen. It is speculated that the lack of cholesterol availability reduces the number of secreted apoB-containing lipoprotein particles by limiting translocation of apoB into the endoplasmic reticulum lumen.