Shu-Ping Hui

Unique character and metabolism of high density lipoprotein (HDL) in fetus

Lipid and lipoprotein profiles, and enzymes for the lipid metabolism were compared between cord and adult blood. Consistent with previous reports, the major lipoprotein in cord blood was high-density lipoprotein (HDL), and that in adult blood was low-density lipoprotein (LDL). The level of apolipoprotein E (apo E) in cord blood was almost equivalent to that in adult blood, while other apolipoproteins and lipids were all lower than the adult level. In cord blood, apo E-rich HDL cholesterol represented more than 30% of total HDL cholesterol (around 11% in adult), and the concentration was about twice of that in adult blood. This apo E-rich HDL cholesterol was poorly esterified (E/T 56%) compared with that in adults (93%). The lecithin:cholesterol acyltransferase (LCAT) activity in cord blood was extremely low, while the activity and mass of cholesteryl ester transfer protein (CETP) were higher than those in adult blood. The apo E genotype did not show influences on total cholesterol, LDL cholesterol, total HDL cholesterol, and apo E rich HDL cholesterol levels in cord blood, as opposed to those in adult blood. The association of D442G mutation of the CETP gene with the increased HDL cholesterol in adult blood was not seen in cord blood. Rather, the mutation was associated with low total cholesterol and LDL cholesterol levels in cord blood. These results indicate that, in fetus, the character and metabolism of HDL, especially of apo E-rich HDL cholesterol, are distinct from those in adults.

Human CD36 deficiency is associated with elevation in low-density lipoprotein-cholesterol.

To find out whether CD36 plays a role in the human lipoprotein metabolism, we studied lipoprotein profiles in subjects with CD36 deficiency. Apparently healthy Japanese volunteers (n = 790) were classified by flow cytometry into three groups of normal (platelet and monocyte CD36+, n = 741, 93.8%), type-II deficiency (platelet CD36- and monocyte CD36+, n = 45, 5.7%), and type-I deficiency (platelet and monocyte CD36-, n = 4, 0.5%). At least one of reported mutations in the CD36 gene was found in all four subjects with type-I deficiency and in 23 of the 45 subjects with type II. Among 779 subjects (731 normals, 44 type II, and four type I) with serum triglyceride levels of <400 mg/dL, serum total cholesterol and low-density lipoprotein (LDL) cholesterol were significantly elevated in type-II deficiency (P = 0.0095 and 0.0382 versus normal, respectively, Scheffes F-test), while differences were not significant in triglyceride and high-density lipoprotein-cholesterol. Similar tendency was observed in type-I deficiency, although the differences were not statistically significant because of small sample size. We conclude that CD36 deficiency elevates LDL cholesterol, indicating a contribution of CD36 to LDL metabolism.

Measurement of lipoprotein particle sizes using dynamic light scattering

Background A simple method for the measurement of LDL particle sizes is needed in clinical laboratories because a predominance of small, dense LDL (sd LDL) has been associated with coronary heart disease. We applied dynamic light scattering (DLS) to measure lipoprotein particle sizes, with special reference to sd LDL. Methods Human serum lipoproteins isolated by a combination of ultracentrifugation and gel chromatography, or by sequential ultracentrifugation, were measured for particle size using DLS. Results The sizes of polystyrene beads, with diameters of 21 and 28 nm according to the manufacturer, were determined by DLS as 19.3 ± 1.0 nm (mean ± SD, n = 11) and 25.5 ± 1.0 nm, respectively. The coefficients of variation for the 21 and 28 nm beads were 5.1% and 3.8% (within-run, n = 11), and 2.9% and 6.2% (between-run, n = 3), respectively. The lipoprotein sizes determined by DLS for lipoprotein fractions isolated by chromatography were consistent with the elution profile. Whole serum, four isolated lipoprotein fractions (CM + VLDL + IDL, large LDL, sd LDL and HDL) and a non-lipoprotein fraction isolated by sequential ultracentrifugation were determined by DLS to be 13.1 ± 7.5, 37.0 ± 5.2, 21.5 ± 0.8, 20.3 ± 1.1, 8.6 ± 1.5 and 8.8 ± 2.0 nm, respectively. Conclusions The proposed DLS method can differentiate the sizes of isolated lipoprotein particles, including large LDL and sd LDL, and might be used in clinical laboratories in combination with convenient lipoprotein separation.

Isolation and characterization of a phenolic antioxidant from the Pacific oyster (Crassostrea gigas).

Using an oxygen radical absorbance capacity (ORAC) assay, antioxidant activity was detected in the ethanol extract of the Pacific oyster, which was purified by sequential extraction with organic solvents. The ethyl acetate fraction showed the strongest antioxidant activity and was further purified, yielding a single compound [as assessed by thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (HPLC)]. This compound was identified as 3,5-dihydroxy-4-methoxybenzyl alcohol on the basis of (1)H and (13)C nuclear magnetic resonance (NMR), heteronuclear multiple-bond correlation (HMBC), and electrospray ionization-mass spectrometry (ESI-MS) spectral analyses, a conclusion that was confirmed by chemical synthesis. The concentration of the compound was 6.7 mg/100 g of whole oyster meat wet weight. This amphiphilic antioxidant retarded the copper-mediated oxidation of low-density lipoproteins (LDLs) and the generation of thiobarbituric acid reactive substances. Furthermore, the compound showed substantial antioxidant activity using the ORAC and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays compared to natural antioxidants. Although the same compound was previously found in brown algae, its presence in other organisms and antioxidant activity are reported here for the first time.

A phenolic antioxidant from the Pacific oyster (Crassostrea gigas) inhibits oxidation of cultured human hepatocytes mediated by diphenyl-1-pyrenylphosphine.

3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA), an antioxidant isolated from the Pacific oyster (Crassostrea gigas), was studied in a cell-based fluorometric antioxidant assay using human hepatocyte-derived cells (C3A) and diphenyl-1-pyrenylphosphine (DPPP) as a fluorescent probe. In comparison with two hydrophilic antioxidants, DHMBA showed the stronger inhibition of DPPP-mediated fluorescence than chlorogenic acid and l-ascorbic acid: at a concentration of 320 μM of DPPP, the inhibition was 26.4±2.6%, 11.1±1.2%, and 0±2.0% for DHMBA, chlorogenic acid, and l-ascorbic acid, respectively (mean±SD, n=4). Their relative oxygen radical absorbance capacities (ORAC) were dissociated with their cell-based antioxidant activities: 1.47±0.40, 4.57±0.30, and 0.53±0.13 μmol TE/μmol for DHMBA, chlorogenic acid, and l-ascorbic acid, respectively (mean±SD, n=4). The amphiphilicity of DHMBA was better than chlorogenic acid and l-ascorbic acid might underlie this dissociation. Since the C3A cells are human hepatoma-derived cells, DHMBA might be useful in the prevention and treatment of liver diseases by involving an oxidation process.

Serum choline plasmalogens, particularly those with oleic acid in sn-2, are associated with proatherogenic state

Serum plasmalogens (Pls) (1-O-alk-1’-enyl-2-acyl glycerophospholipids) are of particular interest for studies on metabolic disorders associated with oxidative stress and chronic inflammation. Serum levels of Pls are known to correlate positively with HDL-cholesterol (HDL-C); however, few studies have examined serum Pls molecular species in association with pathophysiological conditions and their clinical significance. To clarify these, we determined serum levels of individual ether glycerophospholipids in Japanese asymptomatic cohorts (n = 428; 362 male and 66 female subjects) by LC/MS/MS, and examined their correlations with clinical parameters. We found that the proportion of choline Pls (PlsCho) among total serum phospholipids was significantly lower in the male group over 40 years old and was associated with multiple risk parameters more strongly than HDL-C. The abundance of serum PlsCho with oleic acid (18:1) in sn-2 exhibited the strongest positive correlation with serum concentrations of adiponectin and HDL-C, while being inversely associated with waist circumference and the serum levels of TG and small dense LDL-cholesterol. The characterization of serum ether glycerophospholipids verified the specificity of PlsCho, particularly the ones with 18:1 in sn-2, as a sensitive biomarker for the atherogenic state.

Improved HPLC assay for lipid peroxides in human plasma using the internal standard of hydroperoxide.

We have developed a sensitive reversed-phase chemiluminescence HPLC approach for simultaneous quantitative and qualitative analyses of hydroperoxides of cholesteryl ester and TC in human plasma. Standard hydroperoxides of cholesteryl ester and TG and a novel internal standard (1-tetradecanyl 3-octadecenoyloxy-5β-cholan-24-oate monohydroperoxide) (I.S.) were chemically synthesized and the standard curves confirmed to be linear throughout the calibration range (1–1000 pmol). Within-day and between-day CV were less than 7%, and the recoveries were within the range of 84–93%. With sample size minimized to 0.1 mL of plasma for each run, plasma cholesteryl ester hydroperoxide levels were 189±87 nM (mean±SD) in healthy young (22–25 yr old; n=15, male/female=6∶9) and 210 ±69 nM in healthy elderly (39–60 yr old; n=6, male/female= 3∶3). TG hydroperoxide was not detected in healthy subjects. In patients with advanced liver failure (36–67 yr old; n=4, male/female=2∶2), hydroperoxide levels of plasma cholesteryl ester and TG were 11,903±9,553 nM and 3,318±1,590 nM, respectively, indicating an involvement of lipid oxidation. Sensitive and specific monitoring of plasma lipid peroxides using the present chemiluminescence HPLC approach with the synthesized I.S. may help our understanding of chemical and pathophysiological aspects of lipid peroxidation.

Analyses for phosphatidylcholine hydroperoxides by LC/MS.

A new liquid chromatography mass spectrometry (LC/MS) method has been developed for the qualitative and quantitative analyses of phosphatidylcholine hydroperoxides (PC-OOH) in human plasma using a synthetic hydroperoxide (1-stearoyl-2-erucoyl-PC monohydroperoxide, PC 18:0/22:1-OOH) as an internal standard. 1-Stearoyl-2-linoleoyl-PC monohydroperoxide (PC 18:0/18:2-OOH) was identified in plasma by LC/MS by comparison with an authentic standard. The calibration curves obtained for 1-palmitoyl-2-linoleoyl-PC monohydroperoxide, PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were linear throughout the calibration range (0.1-1.0 pmol). The limit of detection (LOD) (S/N=3:1) was 0.01 pmol, and the limit of quantification (LOQ) (S/N=6:1) was 0.1 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 89 and 32 nM, respectively, in a healthy volunteer.

Evaluation of G-to-A substitution in the apolipoprotein A-I gene promoter as a determinant of high-density lipoprotein cholesterol level in subjects with and without cholesteryl ester transfer protein deficiency

The effect of a polymorphism, guanine (G) to adenine (A) substitution in the promoter of apolipoprotein A-I gene at a position 78 bp upstream of the transcription initiation site, on the serum high-density lipoprotein (HDL)-cholesterol level was studied in 168 Japanese subjects with HDL-cholesterol levels ranging from 26 to 171 mg/dl. Considering the significant effect of cholesteryl ester transfer protein (CETP) on the HDL-cholesterol level and the common occurrence of its deficiency, we performed statistical analyses separately for two groups: one without CETP deficiency (n=126) and the other with CETP deficiency (n=42). In the group without CETP deficiency, in which the numbers of G/G, G/A, and A/A genotypes were 92 (73.0%), 28 (22.2%), and 6 (4.8%), respectively, the frequency of the A allele in the subjects with HDL-cholesterol levels of ≥70 mg/dl did not differ from subjects with HDL-cholesterol levels of ≤69 mg/dl, irrespective of gender: 0.154 and 0.145 in males, and 0.182 and 0.174 in females, respectively, for the ≥70 mg/dl and ≤69 mg/dl groups. Additionally, the HDL-cholesterol levels for the subjects with the G/G genotype did not differ from those for the subjects with the A allele: 64 ±22, 58±14, 77±14 and 62±16 mg/dl, respectively, for the G/G, G/A, A/A, and G/A+A/A in males, and 72 ±18, 74±24, 63±4, and 73±23 mg/dl in females. For the group with CETP deficiency, in which the numbers of G/G and G/A+A/A genotypes were 25 (59.5%) and 17 (40.5%), the HDL-cholesterol levels also did not differ: 98±24 mg/dl and 99±30 mg/dl, respectively, for the G/G and G/A+A/A genotypes. Thus, there is no evidence that the polymorphism has any effect on serum HDL-cholesterol levels regardless of CETP status. We conclude that the G-to-A substitution in the promoter of apolipoprotein A-I gene does not significantly alter serum HDL-cholesterol level.

Simple chemical syntheses of TAG monohydroperoxides

For the purpose of synthesizing standards to be used in the quantification of TAG hydroperoxides, three TAG (1,2-dioleoyl-3-palmitoylglycerol, 1-oleoyl-2-linoleoyl-3-palmitoylglycerol, and triolein) monohydroperoxides were chemically synthesized as authentic specimens. TAG were prepared by using a simple condensation in pyridine of glycerol and the corresponding acid chlorides. These TAG were then converted into monohydroperoxides by a photosensitized peroxidation. The synthesized monohydroperoxides were analyzed by normal-phase and RP-HPLC. The results of normal-phase HPLC analysis showed that monohydroperoxides from a corresponding TAG were a mixture of regioisomers. In RP-HPLC, however, the regioisomers of monohydroperoxides were not separated and gave a single peak, which may improve the sensitivity for the detection of TAG monohydroperoxides. In this study TAG monohydroperoxide standards were synthesized; these will be useful for the study of yet unknown biological and pathological roles of TAG hydroperoxides.