Optimized sensitive and inexpensive method to measure D-lactate as a surrogate marker of methylglyoxal fluxes in metabolically relevant contexts.

INTRODUCTION: Plasma D-lactate levels can be considered a surrogate indicator of MG flux. There are commercial methods specifically designed to cover pathological ranges (mmol/l in sepsis or leaky gut) that are not suitable to encompass the metabolically relevant ranges. MATERIAL AND METHODS: We modified and optimized the D-lactate Colorimetric Assay kit MAK058 from Sigma adding an ultracentrifugation step, kinetic reading and increasing reaction temperature. RESULTS AND CONCLUSIONS: We present a modified, optimized and validated method to measure serum D-lactate using a commercial kit to increase the sensitivity of the method to 2 orders of magnitude lower as well as minimizing interferences so that it may be used by researchers to explore the D-lactate pathway in metabolic disorders at a low cost (colorimetric method and a plate reader) and with a much higher specificity.

Reduction of small dense LDL and Il-6 after intervention with Plantago psyllium in adolescents with obesity: a parallel, double blind, randomized clinical trial.

Obesity can lead children and adolescents to an increased cardiovascular disease (CVD) risk. A diet supplemented with Plantago psyllium has been shown to be effective in reducing LDL-C and IL-6 in adolescents. However, there are no studies that have explored small-dense LDL (sdLDL) or HDL subclasses. The aim of this study was to evaluate the impact of a fiber dietary intervention on LDL and HDL subclasses in adolescents with obesity. In this parallel, double blind, randomized clinical trial, the participants were assigned to Plantago psyllium or placebo (10g/day for 7 weeks). We randomized 113 participants, and evaluated and analyzed 100 adolescents (50 in each group), 15 to 19 years with a body mass index of 29-34. We measured biochemical markers LDL and HDL subclasses using the Lipoprint system (Quantimetrix) and IL-6 by ELISA. Post-treatment there was a decrease in sdLDL between the groups 2.0 (0-5.0) vs 1 (0-3.0) mg/dl (p = 0.004), IL-6 median 3.32 (1.24-5.96) vs 1.76 (0.54-3.28) pg/ml, p <0.0001. There were no differences in HDL subclasses and no adverse effects were reported in either group.Conclusions: Small dense LDL and IL-6 reduced in adolescents with obesity when consuming Plantago psyllium. This may be an early good strategy for the reduction of cardiovascular disease risk in this vulnerable population.Trial registration: ISRCTN # 14180431. Date assigned 24/08/2020 What is Known: • Supplementing the diet with Plantago psyllium lowers LDL-C levels. What is New: • First evidence that soluble fiber supplementation like Plantago psyllium decreases small dense LDL particles in association with lowered IL-6, reducing the risk of cardiovascular disease in obese adolescents.

Separation of postprandial lipoproteins: improved purification of chylomicrons using an ApoB100 immunoaffinity method.

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90-94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.

Higher D-lactate levels are associated with higher prevalence of small dense low-density lipoprotein in obese adolescents.

BACKGROUND: Childhood obesity is associated with insulin resistance (IR), increased levels of small dense low-density lipoprotein (sd-LDL) as well as with augmented hepatic de novo lipogenesis, which implies increased triose phosphate fluxes that may lead to increased methylglyoxal (MG) and its catabolic end product D-lactate. We hypothesized that obese adolescents have increased D-lactate serum levels associated with high incidence of sd-LDL. METHODS: This is a cross-sectional study where the anthropometric characteristics, atherogenic dyslipidemia complex, sd-LDL (Lipoprint, Quantimetrix) and D-lactate (kinetic enzymatic analysis) were explored in 30 lean vs. 30 obese adolescents (16 females and 14 males per group) without metabolic syndrome (MetS). Endothelial function by flow-mediated dilation (FMD, by ultrasound) and arterial lesion by carotid intima media thickness (CIMT, by ultrasound) were also measured. RESULTS: The mean age of participants was 16.8 ± 1.4 years. Obese adolescents had a body mass index of 32.7 ± 3.8 vs. 21.8 ± 2.1 in lean participants. The obesity group showed higher D-lactate levels: 6.2 ± 3.0 vs. 4.5 ± 2.5 µmol/L, higher levels of insulin: 15 (9.6-23.5) vs. 7.9 (6.5-10.5) µIU/mL; triglyceride (TG): 1.46 (1.1-1.8) vs. 0.84 (0.6-1.2) mmol/L; non-high-density lipoprotein-cholesterol (NON-HDL-C): 2.8 ± 0.9 vs. 2.3 ± 0.7 mmol/L; total cholesterol (TC)/HDL-C) index: 2.9 ± 0.7 vs. 2.4 ± 0.5; TG/HDL-C index: 2.2 (1.5-2.8) vs. 1.1 (0.8-1.8); %LDL-3: 4.2 ± 4.07 vs. 1.9 ± 2.7; smaller LDL size: 270.6 ± 3 vs. 272.2 ± 1.1 Å. D-lactate correlated positively with LDL-2: r = 0.44 and LDL-3 (sd-LDL): r = 0.49 and negatively with large LDL-1: r = -0.48 and LDL size: r = -0.46; (p<0.05, p<0.01, p<0.001 and p<0.0001, respectively). Obese adolescents showed higher CIMT: 0.51 ± 0.08 vs. 0.46 ± 0.08 mm and lower FMD: 20.3% ± 6.7% vs. 26.0% ± 9.3%. CONCLUSIONS: Obese adolescents display subclinical signs of IR and endothelial dysfunction. Higher serum sd-LDL levels correlated positively with D-lactate levels. These findings suggest an association between atherogenic dyslipoproteinemia and whole body MG fluxes already detectable in apparently healthy obese adolescents.

Correlation of ischemia-modified albumin with SOFA and APACHE II scores in preoperative patients with colorectal cancer.

PURPOSE: Critical illnesses are assessed according to the sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation (APACHE) II. Circulating ischemia-modified albumin (IMA) is a biomarker generated under ischemic and oxidative conditions and may reflect disease severity in preoperative patients. This study investigated the correlations of IMA with SOFA and APACHE II scores in inpatients admitted for colorectal surgery. METHODS: We examined 27 patients with advanced colorectal cancers (mean age 69 years, men/women=15/12). Correlations between SOFA and APACHE II scores in addition to preoperative serum IMA and C-reactive protein (CRP) levels were analyzed. RESULTS: The mean IMA level was 0.5 AU, and the median CRP level was 0.6 mg/dL. Median scores for SOFA and APACHE II were 2 and 12 points, respectively. Significant positive correlations between IMA and SOFA (r=0.45, P<0.05) and IMA and APACHE II (r=0.45, P<0.05) were identified which remained significant in confounder-adjusted analyses. In contrast, weak correlations were observed between CRP and the SOFA and APACHE II scores. CONCLUSIONS: The positive correlations between IMA and both SOFA and APACHE II scores suggest that serum IMA measurements reflect the severity of systemic failure in patients admitted for colorectal surgery in the preoperative phase.

Lean adolescents with insulin resistance display higher angiopoietin like protein 3, ApoC-III and chylomicron remnant dyslipidemia.

BACKGROUND: Triglyceride-rich lipoproteins (TRL: chylomicrons and VLDL) are a key component of diabetes dyslipoproteinemia and cardiovascular risk. We have shown that it is already prevalent in obese adolescents in association with lipoprotein lipase (LPL) dysregulation. Insulin resistance (IR) suffices to produce TRL dyslipoproteinemia and LPL dysfunction even in the absence of obesity. METHODS: This cross-sectional study included euglycemic adolescents between 15 and 19 y, classified in 4 groups according to BMI, HOMA-IR and fasting lipid as: metabolically healthy lean (MHL, n = 30), metabolically unhealthy lean (MUL, n = 25), metabolically healthy obese (MHO, = 30), and metabolically unhealthy obese (MUO, n = 42). RESULTS: As compared to MHL, MUL participants showed 73% higher concentrations of ApoB-48; 84% of ApoC-III; 24% ANGPTL-3; 200% of TG; 218% of VLDL-C and 238% of TG/HDL-C c, No changes were found in LPL mass. Interestingly, the differences in these parameters between MUL and MHO were not significant. CONCLUSION: Euglycemic lean adolescents with IR display TRL dyslipoproteinemia with increased inhibition of LPL as highlighted by higher concentrations of ANGPTL-3, ApoC-III and fasting chylomicron remnants (ApoB-48).

Serum Fatty Acid-Binding Protein 4 Levels in Adolescents: Effect of Insulin Resistance.

Background: Fatty acid-binding protein 4 (FABP4) is an adipokine that plays a causative role in obesity and diabetes. In a stratified cross-sectional study with adolescents, we explored whether changes in FABP4 are already present in lean adolescents, provided they display elements of insulin resistance (IR). Methods: Adolescents were divided in four groups according to body mass index and homeostasis model assessment-IR. Results: In metabolically unhealthy lean (MUL) adolescents (MUL, lean with IR), FABP4 was 33% higher than in healthy counterparts (metabolically healthy lean [MHL]). Obese adolescents without IR (metabolically healthy obesity [MHO]) had 50% higher levels of FABP4 than their lean counterparts (MHL), while levels of FABP4 in obese adolescents with IR (metabolically unhealthy obese [MUO]) were 220% higher than those of MUL adolescents. The differences were significant at least with P < 0.005. MUO > MHO > MUL. Our data demonstrate that the known FABP4 defect in adults with obesity also occurs in youth and even in lean adolescents, suggesting an early association between impaired glucose metabolism and FABP4 irrespective of body weight. FABP4 was more sensitive in discerning each of our 4 subgroups than either adiponectin or leptin. Moreover, evidence for a putative early adiponectin resistance in MUL suggests a combined defect in these adolescents that call for early detection and prevention of the metabolic disturbance that should stay away from concentrating only in subjects with obesity. Conclusions: Our data may serve to draw the considerable attention that is currently paid to FABP4 to the adolescent population, irrespective of the presence of obesity. Further studies with larger cohorts and analyses of visceral and liver fat are warranted.

Serum paraoxonase 1 (PON1) lactonase activity is lower in end-stage renal disease patients than in healthy control subjects and increases after hemodialysis.

BACKGROUND: The mechanism of paraoxonase 1 (PON1) atheroprotective remains elusive. The lactonizing/lactonase activity of PON1 is gaining favor as the most significant in physiology. METHODS: We studied 42 end-stage renal disease (ESRD) patients undergoing hemodialysis (HD) and 49 control subjects. We measured PON1 lactonase, arylesterase and triesterase activities by kinetic methods. RESULTS: Serum lactonase activity was 11% lower in ESRD patients (p<0.0001) and did not correlate with high-density lipoprotein (HDL) cholesterol when controlling for confounders. Lactonase activity was significantly higher after dialysis. Using a repeated measure-ANOVA adjusted for the confounders (age, gender, total cholesterol, triglyceride and HDL cholesterol) we show that the changes in lactonase after dialysis were significant (p<0.0001). HD increases lactonase activity to levels indistinguishable from those of control subjects. In simple linear regression analyses we showed a significant inverse correlation between changes in lactonase and those of creatinine by dialysis (r=-0.339, p=0.028). CONCLUSIONS: ESRD patients maybe more susceptible to lipid peroxidation and to protein homocysteinylation than healthy subjects due to the decreased activity of lactonase. A lower serum lactonase activity would be coupled with delayed catabolism of oxidized phospholipids in low-density lipoprotein and oxidized macrophages, and with greater protein homocysteinylation, accelerating atherogenesis. One mechanism for lower lactonase activity in ESRD patients may be inhibition by uremic toxins and oxidative stress. The pathophysiology of reduced lactonase activity in uremia and the beneficial effects of HD need further investigation.

Higher Hepcidin Levels in Adolescents with Obesity Are Associated with Metabolic Syndrome Dyslipidemia and Visceral Fat.

Tightly regulated iron metabolism prevents oxidative stress. Hepcidin is a hormone that regulates iron flow in plasma; its production is induced by an iron overload and by inflammation. It inhibits iron entry into the circulation by blocking dietary absorption in the duodenum, the release of recycled iron from macrophages and the exit of stored iron from hepatocytes. Varied signals responding to iron stores, erythropoietic activity and host defense converge to regulate hepcidin production and thereby affect iron homeostasis. Although it is known that hepcidin increases when interleukin 6 (IL-6) increases, the relationship between hepcidin, dyslipidemia, insulin resistance (IR) and visceral adiposity index (VAI) in adolescents with obesity is unclear. In this cross-sectional study of 29 obese adolescents and 30 control subjects, we explored the difference of hepcidin, iron metabolism markers and IL-6 between obese and non-obese adolescents, and identified associations with inflammation, atherogenic dyslipidemia and IR. As compared to lean controls, obese participants showed 67% higher hepcidin: 14,070.8 ± 7213.5 vs. 8419.1 ± 4826.1 pg/mLc; 70% higher ferritin: 94.4 ± 82.4 vs. 55.1 ± 39.6 pg/mLa and 120% higher IL-6: 2.0 (1.1-4.9) vs. 0.9 (0.5-1.3) pg/mLd. Transferrin, soluble transferrin receptor and total body iron (as measured by sTFR/ferritin, log10 sTFR/ferritin ratio and sTFR/log ferritin ratios) were not different between the two cohorts. In the whole cohort, hepcidin correlated with VAI (r = 0.29a), sd-LDL (r = 0.31b), HOMA-IR (r = 0.29a) and IL-6 (r = 0.35c). In obese adolescents hepcidin correlated with TG (r = 0.47b), VLDL-C (r = 0.43b) and smaller LDL2 (r = 0.39a). Hepcidin elevation in adolescents with obesity is linked more to inflammation and metabolic alterations than to iron metabolism since the other markers of iron metabolism were not different between groups, except for ferritin. Studies addressing the long-term effects of higher hepcidin levels and their impact on subclinical anemia and iron status are warranted. a p < 0.05; b p < 0.01, c p < 0.001 dp < 0.0001.

Serum cholesterol concentration associated with aspirin esterase activity in older people: preliminary data.

OBJECTIVE: Metabolism of aspirin (acetylsalicylic acid), commonly used in older people for the prevention of cardiovascular disease, is important to the effectiveness of this drug. Whereas part of aspirin hydrolysis occurs in blood, there is a paucity of information in regards to circulating aspirin esterase activity in various physiological and pathological conditions. High aspirin esterase activity, corresponding to faster aspirin hydrolysis (thus aspirin non-responsiveness), may occur in cardiovascular disease-prone states. The objective of this study was to investigate the effects of cardio-metabolic variables such as cholesterol on serum aspirin esterase activity in older people who participated in an intervention study on physical activity. METHODS: A total of 18 non-medicated subjects (7 men/11 women, mean age 67.8 years, body mass index = 23.4 +/- 3.3 kg/m(2)), who completed a 3-month interventional program for a mild-to-moderate increase in physical activity, were analyzed. The body mass index, plasma glucose, serum total cholesterol and aspirin esterase activity were measured in the pre- and post-interventional phases of the study. RESULTS: During the interventional period, the changes in aspirin esterase activity correlated significantly and positively with those of total cholesterol concentrations (r = 0.542, P = 0.020; beta = 0.609, P = 0.035 in a multiple linear regression analysis after adjusting for all the measured variables). CONCLUSION: The results suggest that cholesterol metabolism alterations may be associated with aspirin metabolism in older people.

Higher ANGPTL3, apoC-III, and apoB48 dyslipidemia, and lower lipoprotein lipase concentrations are associated with dysfunctional visceral fat in adolescents with obesity.

BACKGROUND: We hypothesized that adolescents with obesity have higher remnant B48 concentrations associated with lipoprotein lipase dysregulation. METHODS: Cross-sectional study of 32 adolescents with obesity and 27 control subjects. RESULTS: As compared to lean controls, obese participants showed 35% higher concentrations of apoB48: 3.60 (2.93-4.30) vs 2.65 (1.64-3.68) ng/ml; 28% of apoC-III: (72.7 (58.6-89.7) vs 56.9 (44.8-79.8 ug/ml and 17% ANGPTL 3: (72.2 ± 20.2 vs 61.2 ± 19.2 ng/ml). This was accompanied by a 33% reduction in LPL: 13.1 ± 5.1 vs 18.9 ± 4.7 ng/ml. Obese participants had 25% lower adiponectin 2.9 (1.9-3.8) vs 4.4 (3.2.-5.2) µg/ml; 260% higher leptin 25.7 (11.2-44.8) vs 9.3 (2.8-20.7) ng/ml c and 83% higher Il-6: 2.2 (1.3-5.4) vs 1.2 (0.8-1.4) pg/ml. ApoC-III and ANGPTL3 correlated positively with VAI; ANGPTL3 negatively with HDL-C; LDL size and VLDL-C. ApoB48 correlated negatively with LDL-C. CONCLUSIONS: Adolescents with obesity show higher ANGPTL3 compounded with increased apoC-III associated with increased CR and lower LPL mass. This is associated with inflammation and visceral fat. The significance of these findings resides in that they shed light on a mechanism for TRL dyslipidemia in adolescents: increased LPL inhibition impairs VLDL and chylomicron catabolism leading to atherogenic remnants.

Paraoxonase 1, HDL Subclasses and Post Surgery Acute Inflammation: A Pilot Study.

High density lipoproteins (HDL) structure and function studies are needed to better understand the heterogeneous nature of the HDL particle, and its interaction with associated proteins such as apolipoprotein A-1 (ApoA-1), paraoxonase 1 (PON1) and the environment. Our study assesses the effects of acute inflammation on PON1 and HDL subclasses in post-surgical colorectal cancer patients. PON1 was measured kinetically through its arylesterase and lactonase activity and HDL sub-classes were measured using Quantimetrix Lipoprint® System. White blood cells (WBC) counts, c-reactive protein (CRP) and serum amyloid A (SAA) levels were also analyzed using standard techniques. Our findings show that baseline PON1 activity is lower in colorectal cancer patients and significant reductions are observed in the acute inflammatory state post-surgery. PON1 changes are also inversely related to inflammatory markers such as SAA and CRP. In addition, our preliminary findings show that small and intermediate HDL decreases post-op Day 1. In conclusion, our study demonstrates the effects of chronic and acute inflammation on PON1. Specifically, PON1 arylesterase and lactonase activity is lower in states of chronic inflammation and further decreased in the acute inflammatory state. Additionally, in our limited sample size, while changes in PON1 and HDL subclasses may be variable in the acute inflammatory period, small HDL decreased with a loss of PON1 activity in the subacute phase.

Isocaloric Fructose Restriction Reduces Serum d-Lactate Concentration in Children With Obesity and Metabolic Syndrome.

OBJECTIVE: To investigate the link between dietary sugar consumption and two separate pathogenetic mechanisms associated with metabolic syndrome: de novo lipogenesis (DNL) and nonenzymatic glycation. DESIGN AND PARTICIPANTS: We assessed changes in serum d-lactate (the detoxification end-product of methylglyoxal) concentration in response to 9 days of isocaloric fructose restriction in 20 children with obesity and metabolic syndrome, and examined correlations with changes in DNL, liver fat, insulin sensitivity, and other metrics of hepatic metabolism. INTERVENTIONS: Nine days of dietary sugar restriction, with substitution of equal amounts of refined starch. MAIN OUTCOME MEASURES: On days 0 and 10, children had laboratory evaluation of d-lactate levels and other analytes, and underwent oral glucose tolerance testing, magnetic resonance spectroscopy to quantify fat depots, and 13C-acetate incorporation into triglyceride (TG) to measure DNL. RESULTS: d-Lactate was associated with baseline liver fat fraction (P < 0.001) and visceral adipose tissue (P < 0.001) but not with subcutaneous adipose tissue. At baseline, d-lactate was positively correlated with DNL-area under the curve (AUC) (P = 0.003), liver fat fraction (P = 0.02), TG (P = 0.004), and TG/high-density lipoprotein ratio (P = 0.002). After 9 days of isocaloric fructose restriction, serum d-lactate levels reduced by 50% (P < 0.0001), and changes in d-lactate correlated with both changes in DNL-AUC and measures of insulin sensitivity. CONCLUSION: Baseline correlation of d-lactate with DNL and measures of insulin sensitivity and reduction in d-lactate after 9 days of isocaloric fructose restriction suggest that DNL and nonenzymatic glycation are functionally linked via intermediary glycolysis in the pathogenesis of metabolic syndrome and point to fructose as a key dietary substrate that drives both pathways.

Soluble Receptor for Advanced Glycation End Products and Its Correlation with Vascular Damage in Adolescents with Obesity.

OBJECTIVE: The aim of this study was to evaluate soluble receptor for advanced glycation end products (sRAGE) and advanced glycation end products (AGEs) in adolescents with and without obesity (OB) and their correlation with vascular damage. METHODS: This is a cross-sectional study with 15-19 years old adolescents: 33 with OB and 33 with normal weight (NW), each group included 17 male and 16 female. Lipid profile, insulin, carboxymethylysine (CML), sRAGE, total AGEs, and dietary AGEs intake (dAGEs) were evaluated. Vascular damage was measured by flow-mediated vasodilation (FMD) and arterial stiffness index (Iß). Homeostatic model assessment-insulin (HOMA-IR) and atherogenic index (AI) were calculated. RESULTS: The group with OB had higher triglycerides (TG; p < 0.0001), AI (p < 0.001), HOMA-IR (p < 0.0001), dAGEs intake (p < 0.0001), lower CML (p = 0.05), total AGEs (p < 0.01), sRAGE (p < 0.001), and FMD (p < 0.002). In the total group, sRAGE correlated with AI (r = -0.26 p = 0.037); in the NW group, CML correlated with Iß (r = -0.36; p = 0.037); and in the group of adolescents with OB, sRAGE correlated with FMD (r = -0.37; p = 0.037) and Iß (r = 0.47; p = 0.006), while CML and total AGEs correlated with AI, p = 0.007 and p < 0.01, respectively). CONCLUSIONS: The group of adolescents with OB showed higher cardiometabolic risk as shown by higher TG, AI, HOMA-IR, and lower sRAGE and FMD. sRAGE correlated negatively with FMD and positively with Iß, so it could be suggested as a biochemical marker of impaired endothelial function.

Advanced glycation endproducts form during ovalbumin digestion in the presence of fructose: Inhibition by chlorogenic acid.

BACKGROUND: One mechanism by which fructose could exert deleterious effects is through intestinal formation and absorption of pro-inflammatory advanced glycation endproducts via the Maillard reaction. We employed simulated stomach and duodenum digestion of ovalbumin (OVA) to test the hypothesis that advanced glycation endproducts (AGEs) are formed by fructose during simulated digestion of a ubiquitous food protein under model physiological conditions. METHODS: OVA was subjected to simulated gastric and intestinal digestion using standard models, in presence of fructose or glucose (0-100mM). Peptide fractions were analyzed by fluorescence spectroscopy and intensity at Excitation: λ370nm, Emission: λ 440nm. RESULTS: AGE adducts formed between fructose and OVA, evidenced by the peptide fractions (<5kDa) at times (30min) and concentration ranges (10mM) plausibly found in the intestines, whereas no reaction occurs with glucose. The reaction was inhibited by chlorogenic acid at concentrations compatible with those found in the gut. The reaction was also inhibited by aminoguanidine, a specific antiglycation agent. CONCLUSION: Our study showed fructose-AGE formation on a ubiquitous dietary protein under model physiological conditions. Our study also suggests ways to decrease the damage: enteral fructose-AGE formation may be partially inhibited by co-intake of beverages, fruits and vegetables with concentrations of phenolics high enough to serve as anti-glycation agents.

Postprandial Paraoxonase 1 Activity Following Consumption of Recommended Amounts of Mixed Meals in Healthy Males.

AIM: Postprandial lipid level increases induce oxidative stress, which is involved in atherogenesis. The antioxidant properties of paraoxonase 1 (PON1) have attracted attention. However, changes in postprandial PON1 levels differ across prior studies, and changes in PON1 lactonase activity, potentially relevant to PON1 physiology, after the consumption of ordinary meals are unknown. Herein we evaluated postprandial serum lipid levels and PON1 changes following mixed-meal consumption of the amounts recommended for ordinary meals. METHODS: Nine healthy male volunteers consumed three different meals in a randomized cross-over design. The test meals were as follows: S, white rice; SMF, S with fat-containing protein-rich main dishes; and SMFV: SMF with vegetable dishes. The serum lipid concentrations and PON1 lactonase and arylesterase activities were determined during a three-hour period after the consumption of these meals. RESULTS: The postprandial triglyceride levels were higher after consuming the SMF and SMFV meals than after consuming the S meal. Despite postprandial high-density lipoprotein cholesterol being unchanged, PON1 lactonase activity was decreased, while PON1 arylesterase activity was increased in the postprandial state after all test meals. Postprandial changes in lactonase and arylesterase activities did not differ among the test meals. CONCLUSIONS: Inverse changes in PON1 lactonase and arylesterase activities were observed after consuming recommended ordinary meals. This observation provides useful information for choosing PON1 species as postprandial markers.

Short-term isocaloric fructose restriction lowers apoC-III levels and yields less atherogenic lipoprotein profiles in children with obesity and metabolic syndrome.

BACKGROUND AND AIMS: Dietary fructose may play a role in the pathogenesis of metabolic syndrome (MetS). In a recently published study of obese children with MetS, we showed that isocaloric fructose restriction reduced fasting triglyceride (TG) and LDL-cholesterol (LDL-C). In these ancillary analyses, we tested the hypothesis that these effects were also accompanied by improved quantitative and qualitative changes in LDL and HDL subclasses and their apolipoproteins; as well as change in VLDL, particularly apoC-III. METHODS: Obese children with MetS (n = 37) consumed a diet that matched self-reported macronutrient composition for nine days, with the exception that dietary fructose was reduced from 11.7 ± 4.0% to 3.8 ± 0.5% of daily calories and substituted with glucose (in starch). Participants underwent fasting biochemical analyses on Days 0 and 10. HDL and LDL subclasses were analyzed using the Lipoprint HDL and LDL subfraction analysis systems from Quantimetrix. RESULTS: Significant reductions in apoB (78 ± 24 vs. 66 ± 24 mg/dl) apoC-III (8.7 ± 3.5 vs. 6.5 ± 2.6 mg/dl) and apoE (4.6 ± 2.3 vs. 3.6 ± 1.1 mg/dl), all p < 0.001) were observed. LDL size increased by 0.87 Å (p = 0.008). Small dense LDL was present in 25% of our cohort and decreased by 68% (p = 0.04). Small HDL decreased by 2.7% (p < 0.001) and large HDL increased by 2.4% (p = 0.04). The TG/HDL-C ratio decreased from 3.1 ± 2.5 to 2.4 ± 1.4 (p = 0.02). These changes in fasting lipid profiles correlated with changes in insulin sensitivity. CONCLUSIONS: Isocaloric fructose restriction for 9 days improved lipoprotein markers of CVD risk in children with obesity and MetS. The most dramatic reduction was seen for apoC-III, which has been associated with atherogenic hypertriglyceridemia.

Small-dense low-density lipoproteins are the predominant apoB-100-containing lipoproteins in cord blood.

OBJECTIVES: Low density lipoprotein (LDL)-cholesterol level in cord blood is approximately 30%. The aim of our study was to specifically explore LDL apoB distribution across sizes in cord blood serum. DESIGN AND METHODS: We studied 83 healthy neonates and 17 paired healthy mothers. Plasma glucose and serum lipids, such as low-density lipoprotein (LDL) cholesterol (LDL-C), HDL cholesterol (HDL-C) and triglycerides (TG), were measured using enzymatic methods. Distribution of apoB-100 was performed by western blot and immunodetection on native 4-12% polyacrylamide gels. LDL subclasses were analyzed by Lipoprint-LDL. RESULTS: Neonates show the expected lower content of apoB LDL and small dense LDL is the predominant apoB containing particle: 67 ± 7%. However, only 1.5% is sdLDL by Lipoprint. Maternal serum contains a large proportion of apoB in smaller LDL, 47 ± 6% as compared to non-pregnant women, 6 ± 1%, p<0.001. CONCLUSIONS: Neonates show the expected lower content of apoB-LDL but in an inverse distribution; sdLDL being the predominant particle. This novel finding for apoB sdLDL is consistent with previous data on HPLC studies showing increased middle and small-sized LDL lipid content in neonates as compared to adults which amounts to 84% of total LDL. Comparison of the results with Lipoprint LDL (lipids) with gradient gel electrophoresis native western blot (apoB-100) suggests that neonates carry fractions of small LDL that are comparatively poor in lipids as compared with their mothers. Further studies are warranted on the issue of sdLDL in neonates.

Paraoxonase 1 lactonase activity and distribution in the HDL subclasses in the cord blood.

OBJECTIVES: Paraoxonase 1 (PON1) is a lactonase with important antioxidant and immunoprotective properties. We hypothesized that PON1 lactonase activity, PON1, and high-density lipoprotein (HDL) subclasses distribution are different in neonates than in adults. MATERIAL AND METHODS: We studied 83 healthy term neonates (34 males and 49 females) who were born by spontaneous, uncomplicated vaginal delivery. The study also included 17 paired maternal blood samples as well as 20 non-pregnant women collected for comparison. Total and free PON1 lactonase and arylesterase activity, HDL subclasses, PON1, and apolipoprotein distribution in the subclasses were assayed. RESULTS: PON1 arylesterase activity in the cord blood represented 37% ± 4 of the maternal activity, whereas the PON1 lactonase activity amounted to only 23% ± 5 of the maternal activity. The free arylesterase and lactonase activities were higher in the cord blood by 16 and 36%, respectively. There is a 65% lower HDL2b PON1 in the cord blood than in the maternal serum. When the Lipoprint HDL subclasses were assayed, the neonates showed a larger content (52% higher) of very large HDL as well as a characteristic peak in the middle-sized HDL5 which is unremarkable in the mothers. CONCLUSION: The novel findings of this study are that the neonates have lower PON1 lactonase activity, higher free PON1, different distributions of PON1 in the HDL subclasses as compared with their mother and adults as well as a distinctive HDL subclass lipid profile. Our data also suggest that the neonate HDL is enriched with an intermediate-sized (and/or less charged HDL) that is also rich in active PON1.

Evidence for the presence of active paraoxonase 1 in small-dense low-density lipoprotein.

AIM: We have recently demonstrated the quick ex vivo transfer of paraoxonase 1 (PON1) activity from high-density lipoprotein (HDL) to small, dense low-density lipoprotein (sdLDL). We set out to assess whether sdLDL contains active PON1 in vivo. METHODS: We conducted a nested case-control, proof of principle study with the Japanese healthy subjects with normal lipids (n = 23) and age and gender-paired dyslipidemic subjects (n = 17). Lipid panels, lactonase and arylesterase assays, and PON1 zymogram in the LDL and HDL subclasses were assessed. RESULTS: PON1 specific activity in the high-molecular weight lipoprotein fraction corresponding to LDL migration was found in 48% of normo and in 29% of dyslipidemic Japanese subjects. This band co-localizes with apoB100 and not Lp(a) and displays a lower molecular mass than the bulk of LDL. CONCLUSION: We provide evidence, for the first time, that native sdLDL contains up to 4% of the total PON1 activity in the serum of up to 48% of the Japanese subjects. Could the PON1-containing sdLDL represent a set of particles with a defense mechanism from oxidation and therefore its levels actually prove to be atheroprotective? If further studies confirm this contention, a zymogram of PON1 in LDL subclasses could be a functional assay that complements the current methods that only inform on the size and lipid concentration of these particles.