Gel Permeation High-performance Liquid Chromatography Research Articles

Relationship between the cholesterol and triglyceride content of lipoprotein subclasses and carotid intima-media thickness: A cross-sectional population-based study

BackgroundThe association between lipoprotein subclasses and carotid intima-media thickness (cIMT) progression has yet to be fully evaluated. We assessed which lipoprotein subclasses were associated with maximum cIMT levels in the general population. MethodsIn this study, cholesterol and triglyceride content of 20 lipoprotein subclasses were analyzed using gel permeation high-performance liquid chromatography (GP-HPLC) in 864 Japanese women and men (mean age 57 y, free of chronic liver or kidney diseases and off lipid-lowering, hormone replacement, or adrenocorticosteroid medications). Univariate and multivariate regression analyses and univariate and partial correlation analyses were performed to examine the relationships between lipoprotein subclasses and maximum cIMT levels. ResultsAfter adjusting for age, sex, systolic blood pressure, smoking, diabetes, and anti-hypertensive agents, elevated low-density lipoprotein (LDL)-2 and −3 cholesterol (particle diameter 25.5 nm and 23.0 nm, respectively; medium and small LDL) were associated with higher maximum cIMT levels in both women and men (all p for trend < 0.05). These associations were significant even after participants taking anti-diabetic or anti-hypertensive agents were excluded. No significant associations were found between any triglyceride subclasses and maximum cIMT levels. ConclusionsSmaller LDL particle cholesterol values are the most atherogenic lipoprotein parameter.

The effects of pemafibrate and omega-3 fatty acid ethyl on apoB-48 in dyslipidemic patients treated with statin: A prospective, multicenter, open-label, randomized, parallel group trial in Japan (PROUD48 study).

We compared the lowering effects of pemafibrate and omega-3 fatty acid ethyl on fasting apolipoprotein (apo) B-48 (apoB-48), a marker that reflects postprandial hypertriglyceridemia, which is one of the residual risks for atherosclerotic cardiovascular disease (ASCVD) with statin treatment. This prospective, multicenter, open-label, randomized, parallel group trial was conducted at 4 medical institutions between April 2020 and May 2022. A total of 126 ambulatory patients with dyslipidemia receiving statin treatment for more than 4 weeks, aged 20-79 years with fasting triglyceride (TG) levels of ≥177 mg/dl were randomly assigned to 16-week pemafibrate 0.4 mg per day treatment group (PEMA, n = 63) or omega-3 fatty acid ethyl 4 g per day treatment group (OMEGA-3, n = 63). The primary endpoint was the percentage change in fasting apoB-48 from baseline to week 16. The percentage changes in fasting apoB-48 in PEMA and OMEGA-3 were -50.8% (interquartile range -62.9 to -30.3%) and -17.5% (-38.3 to 15.3%) (P < 0.001), respectively. As the secondary endpoints, the changes in fasting apoB-48 in PEMA and OMEGA-3 were -3.10 μg/ml (-5.63 to -1.87) and -0.90 μg/ml (-2.95 to 0.65) (P < 0.001), respectively. Greater decreases with significant differences in the percentage changes in TG, remnant lipoprotein cholesterol, apoC-III, fasting plasma glucose, alanine aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase were observed in PEMA, compared with OMEGA-3. Greater increases with significant differences in those in high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-II were observed in PEMA, compared with OMEGA-3. PEMA showed anti-atherosclerotic lipoprotein profiles in gel-permeation high-performance liquid chromatography analyses, compared with OMEGA-3. Although adverse events occurred in 9 of 63 (14.3%) patients in PEMA and 3 of 63 (4.8%) patients in OMEGA-3, no serious adverse events associated with drug were observed in either group. This is the first randomized trial to compare the lowering effects of pemafibrate and omega-3 fatty acid ethyl on fasting apoB-48. We concluded that pemafibrate was superior to omega-3 fatty acid ethyl in lowering effect of fasting apoB-48. Pemafibrate is expected to reduce the residual risk for ASCVD with statin treatment. https://rctportal.niph.go.jp/en, identifier jRCTs071200011.

Development of internal standard for lipoprotein subclass analysis using dual detection gel-permeation high-performance liquid chromatography system.

The LipoSEARCH® System is an innovative lipoprotein class analysis method based on gel-permeation high-performance liquid chromatography (HPLC). This system uses a gel permeation column to separate the major lipoprotein subclasses (chylomicron, very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein) in serum according to particle size and splits them into two pathways to measure total cholesterol (TC; esterified + unesterified cholesterol) and triglyceride (TG) concentrations simultaneously to obtain chromatograms for each.These chromatograms were analyzed based on the results of the calibration serum by fitting Gaussian curves to profile the 20 lipoprotein subclasses defined in detail. An important assumption of this HPLC system is its simultaneous detection of two pathways to guarantee the accuracy of each analysis. Therefore, in the present study, we investigated the development of an internal standard that can guarantee the simultaneous detection of this system by adding a pigment to the serum. We focused on quinone pigments with absorption at 550 nm, which is the wavelength used for the enzymatic assay of TC and TG concentrations in the system. As a result, we succeeded in producing overlapping pigment peaks that appeared after the analytical chromatograms in two pathways. It is also suggested that the pigment solution as an internal standard is stable in freezing storage and has little effect on the analysis. The developed internal standard is expected to contribute to the accuracy assurance of lipoprotein analysis by this dual-detection HPLC system.

In vitro angiotensin-converting enzyme and dipeptidyl peptidase-IV inhibitory, and antioxidant activity of blue mussel (Mytilus edulis) byssus collagen hydrolysates

AbstractLarge quantities of mussel byssus are generated annually as a co-product of the mussel-processing industry. This fibrous material is a rich source of collagen, which when extracted has potential uses as an alternative source of collagen for food applications. However, due the complex structure of the material, the extraction of the collagenous components using food-friendly strategies has proved challenging to date. An enzyme-aided method, using a proline endoproteinase, was employed for the extraction of collagen from mussel byssus yielding 138.82 ± 2.25 mg collagen/g dry weight. Hydrolysates of the collagen extract were generated using five food-grade enzyme preparations with Corolase® PP giving the highest extent of hydrolysis. Reversed-phase and gel permeation high-performance liquid chromatography of the extracted collagen and its enzymatic hydrolysates showed significant hydrolysis of collagen. The hydrolysates generated with Corolase® PP showed the highest in vitro bioactivities: angiotensin-converting enzyme (ACE) IC50 = 0.79 ± 0.17 mg/ml, dipeptidyl peptidase-IV (DPP-IV) IC50 = 0.66 ± 0.17 mg/ml and oxygen radical absorbance capacity (ORAC) activity = 311.23 ± 13.41 µmol trolox equivalents (TE)/g. The results presented herein indicate that in addition to acting as an alternative source of collagen for food applications, mussel byssus collagen-derived hydrolysates have potential applications as functional food ingredients for the management of metabolic diseases such as type II diabetes and hypertension.

Abstract 9165: Relationship Between the Cholesterol and Triglyceride Content of Lipoprotein Subclasses and Carotid Imtima-Media Thickness: Results from the Kyushu and Okinawa Population Study

Introduction: The association between 20 lipoprotein cholesterol and triglycerides subclasses and carotid intimal medial thickness (cIMT) progression has not been evaluated fully. Objective: Our goal was to assess which lipoprotein subclasses were associated with max cIMT levels. Methods: Levels of cIMT and 20 lipoprotein cholesterol and triglycerides subclasses using gel permeation high-performance liquid chromatography (GP-HPLC) were analyzed in 864 men and women (mean age 57 years, free of chronic liver or kidney diseases and off cholesterol-lowering, hormone replacement, or adrenocorticosteroid medications). Univariate and multivariate regression analyses were performed to examine the relationships between lipoprotein subclasses and max cIMT levels. Results: Max cIMT levels were 0.90 mm in women and 0.97 mm in men. After adjustment for age, gender, systolic blood pressure, smoking habits, diabetes, and hypertension treatment, elevated low-density lipoprotein cholesterol-2 and -3 were associated with higher max cIMT levels in both women and men. Decreased high-density lipoprotein cholesterol-7 were associated with higher max cIMT levels only in women (all P for trend &lt; 0.05). However, no associations were found between any triglyceride subclasses and max cIMT levels. Conclusions: Our data indicate that elevated smaller LDL cholesterol levels separated by GP-HPLC were significantly associated with max cIMT in both men and women. Our results support the concept that small dense LDL cholesterol is the most atherogenic lipoprotein parameter.

Hypocholesterolemic Effect of Blackcurrant (Ribes nigrum) Extract in Healthy Female Subjects: A Pilot Study.

Blackcurrant extract (BCE) ameliorates dyslipidemia in menopausal model animals and in elderly women at a risk of dyslipidemia. However, it is unknown whether the daily intake of BCE can prevent lipid abnormalities in healthy individuals. Lipids are essential for the body, but they also cause arteriosclerosis. In this noncomparative pilot study, we examined the effects of BCE administered for 29 days on serum lipids in young healthy women. Blood samples were collected before and on days 4 and 29 after BCE intake, and 20 lipoprotein fractions in the serum were separated using a gel-permeation high-performance liquid chromatography method to measure the triacylglycerol and cholesterol levels in lipoproteins. There were no effects on lipids on day 4 of BCE intake, but the total cholesterol level decreased on day 29. Furthermore, the levels of total very-low-density lipoprotein (VLDL) cholesterol, small VLDL cholesterol, and large low-density lipoprotein cholesterol were significantly decreased. These results suggest that the daily intake of BCE has a hypocholesterolemic effect in healthy women, and that it is effective in preventing atherosclerosis.

Chemical structure of a novel heteroglycan polysaccharide isolated from the biomass of Ophiocordyceps Sobolifera

This study aims at identifying the structure of polysaccharide (PS) extracted from the biomass of Vietnam-grown Ophiocordyceps sobolifera for the first time. The molecular weight of the obtained polysaccharide (denoted as PS-T100) is found to be 2.29 × 105 Da using gel permeation high-performance liquid chromatography. The structural investigations of the sample PS-T100 unveil a novel structure composed of a heteropolysaccharide with the appearance of two main sugars, D-glucose and D-mannose, in the backbone. Indeed, methylation analysis indicates that PS-T100 is mainly composed of (1→3)-linked glucosyl and (1→3)-linked mannosyl units. Moreover, 1D and 2D nuclear magnetic resonance spectroscopy interpretations of the sample PS-T100 prove the repeating unit of the isolated polysaccharide is [→3)-β-D-Glcp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→]n. Such a structure appears to be a novel polysaccharide. The obtained results contribute to enriching the database of Ophiocordyceps sobolifera- extracted PSs and open up a new pathway toward the production of PS-T100 for biomedical applications.

Lipoprotein characterization in Quaker parrots (Myiopsitta monachus) using gel‐permeation high‐performance liquid chromatography

Lipid accumulation disorders, such as atherosclerosis and hepatic lipidosis, are common in psittacine birds and associated with various dyslipidemias. Gel-permeation high-performance liquid chromatography (GP-HPLC) is a reference method for advanced lipoprotein profiling based on particle size separation, followed by an analysis of lipid contents. The objectives were to (a) characterize Quaker parrot lipoproteins using a commercial GP-HPLC method (Liposearch panel), and (b) obtain preliminary information on the reliability of the Friedewald formula for low-density lipoprotein-cholesterol (LDL-C) measurements. Plasma samples were collected from 12 fasted healthy Quaker parrots. Cholesterol concentrations, triglyceride concentrations, particle sizes, and particle numbers were determined by GP-HPLC for four classes and 20 sub-fractions of lipoproteins. The LDL-C concentrations obtained using the Friedewald formula and direct measurements were compared with Bland-Altman plots. Alternate formulas were determined using multiple linear regression. High-density lipoprotein (HDL) was the predominant lipoprotein in Quaker parrots, and most particles were of medium-to-small sizes belonging to two sub-fractions (average size, 10.6nm). LDL was the second most common lipoprotein and included large-to-small particles belonging to three sub-fractions (average size, 24.9nm). Very-low-density lipoproteins (VLDL) and portomicrons were present in low concentrations. The Friedewald formula underestimated LDL-C concentrations with a significant bias of 0.44mmol/L. An alternate formula was proposed: LDL-C=0.75*Non-HDL-C. GP-HPLC allowed unprecedented characterization of plasma lipoproteins in Quaker parrots. Characterizing psittacine lipoprotein is useful for validation and interpretation of routine clinical tests as well as for use in epidemiologic and experimental research on psittacine lipid accumulation disorders.

Administration of eicosapentaenoic acid may alter lipoprotein particle heterogeneity in statin-treated patients with stable coronary artery disease: A pilot 6-month randomized study

BackgroundWe hypothesized that the addition of eicosapentaenoic acid (EPA) to ongoing statin therapy could change the particle heterogeneity of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles, even in stable coronary artery disease (CAD) patients. MethodsWe assigned CAD patients already receiving statin therapy to one of two groups: an EPA group (1800 mg/day; n = 30) and a control group (n = 30). A gel permeation high-performance liquid chromatography method was used to measure the particle concentration and number of lipoprotein subclasses. ResultsIn the EPA group, significant decreases of both the concentration and number of medium LDL (p = 0.0002 and 0.0001), small LDL (p = 0.0004 and 0.0005) and very small LDL (p = 0.0005 and 0.002) particles were observed. Conversely, the concentration and number of large HDL particles increased significantly (p = 0.024 and 0.048). The concentration of very large HDL particles also increased significantly (p = 0.028). Furthermore, significant correlations between the variables that showed significant changes in the LDL and HDL particle subclasses, and the EPA/arachidonic acid (AA) ratio were found. No other significant associations of lipoprotein particle heterogeneity with the serum EPA/AA ratio were noted in either the control group or the EPA group. Interestingly, univariate and multivariate regression analyses revealed that increased serum lecithin–cholesterol acyltransferase activity, a key enzyme of HDL cholesterol efflux, was a predictor for increased above-mentioned HDL particles subclasses. ConclusionsAdministration of EPA might alter both LDL and HDL particle heterogeneity, causing decreased concentration and number of smaller LDL particles and increased concentration and number of larger HDL particles. Furthermore, addition of EPA to ongoing statin therapy appears to be capable of increasing the EPA/AA ratio, which might have an anti-atherosclerotic effect on lipoprotein particle heterogeneity, even in stable CAD patients with well-controlled serum lipid levels. Clinical Trial RegistrationUMIN (http://www.umin.ac.jp/) Study ID: UMIN000010452.

Fucoidan Isolated from Saccharina japonica Inhibits LPS-Induced Inflammation in Macrophages via Blocking NF-κB, MAPK and JAK-STAT Pathways.

Fucoidan has been reported to have a variety of biological activities. However, different algae species, extraction methods, harvesting seasons, and growth regions lead to the structural variation of fucoidan, which would affect the bioactivities of fucoidan. To date, the anti-inflammatory properties and the underlying mechanism of fucoidan from brown alga Saccharina japonica (S. japonica) remain limited. The aims of the present study were to investigate the structure, the anti-inflammatory properties, and the potential molecular mechanisms of fucoidan isolated from S. japonica (SF6) against lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. SF6 was characterized using high performance liquid gel permeation chromatography (HPGPC), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR), and observed to be rich in fucose, galactose, and sulfate. Additionally, results showed that SF6 remarkably inhibited LPS-induced production of various inflammatory mediators and pro-inflammation cytokines, including nitric oxide (NO), NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-β (IL-β), and interleukin-6 (IL-6). A mechanism study showed that SF6 could effectively inhibit inflammatory responses through blocking LPS-induced inflammation pathways, including nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase (JAK)-2 and signal transducer and activator of transcription (STAT)-1/3 pathways. These results suggested that SF6 has the potential to be developed as an anti-inflammatory agent applied in functional food.

Separation and Purification of Lipase Inhibitory Peptide from Fermented Milk by Lactobacillus plantarum Q180

In this study, we separated and purified lipase inhibitory peptide from fermented milk by Lactobacillus plantarum Q180 with the aim of developing a new functional anti-lipase activity yogurt product. L. plantarum 180 was inoculated into 10% reconstituted skimmed milk and incubated at 37°C until the pH of the culture reached pH 4.4. The lipase activity was measured using porcine pancreatic lipase. The lipase inhibitory peptides were gradually isolated by ultrafiltration, reversed phase column chromatography (RPC), reversed phase high-performance liquid chromatography (RP-HPLC), and gel permeation high-performance liquid chromatography (GP-HPLC) from the fermented milk by L. plantarum Q180. An ODS-AQ column was used for the RPC, a Vydac C18 column for the RP-HPLC, and a Superdex Peptide HR column for the GP-HPLC. The peptide was composed of Asp, Thr, Ile, Ser, Ala, and Gln, and the anti-lipase activity (IC50) was 2,817 μg/mL.

Optimization extraction, characterization and anticancer activities of polysaccharides from mango pomace

Response surface methodology was used to optimize the extraction conditions for ultrasonic-assisted extraction of polysaccharides from mango pomace. The Optimum extraction conditions consisted of extraction temperature of 74 °C, ultrasonic power of 170 W, extraction time of 100 min, and raw material-to-water ratio of 1:40 g/mL. Under these conditions, the extraction yield was 3.71 ± 0.07%. Three novel polysaccharide fractions, MG-1, MG-2 and MG-3 were purified from the crude polysaccharides by using DEAE-52 cellulose and Sephadex G-100 column chromatography. The molecular weight and monosaccharide composition of polysaccharide fractions (MPFs) were analyzed by high performance liquid gel permeation chromatography (HPGPC) and HPLC analysis, respectively. The characterizations of MPFs were conducted with FT-IR, 1H NMR and SEM. Furthermore, the anticancer activities of the polysaccharide fractions were also investigated in vitro. Results showed that MG-1, MG-2 and MG-3 exhibited significant anticancer activities against HepG2, MCF-7, A549, HeLa, A2780, HCT-116 and BGC-823 cells in a dose-dependent manner. MPFs were also showed to promote apoptosis as seen in the nuclear morphological examination study using calcein acetyl methoxy methyl easter (calcein-AM) and propidium iodide (PI) staining. This research could serve as a theoretical reference for the efficient utilization of MPFs in biomedical and functional food.

Immune-mediated acquired lecithin-cholesterol acyltransferase deficiency: A case report and literature review

Recessive inherited disorder lecithin-cholesterol acyltransferase (LCAT) deficiency causes severe hypocholesterolemia and nephrotic syndrome. Characteristic lipoprotein subfractions have been observed in familial LCAT deficiency (FLD) with renal damage. We described a case of acquired LCAT deficiencies with literature review. The lipoprotein profiles examined by gel permeation-high-performance liquid chromatography (GP-HPLC) and native 2-dimensional electrophoresis before and after prednisolone (PSL) treatment. Here we describe the case of a 67-year-old man with severely low levels of cholesterol. The serum LCAT activity was undetectable, and autoantibodies against it were detected. The patient developed nephrotic syndrome at the age of 70years. Renal biopsy revealed not only membranous glomerulonephritis but also lesions similar to those seen in FLD. We initiated PSL treatment, which resulted in remission of the nephrotic syndrome. In GP-HPLC analysis, lipoprotein profile was similar to that of FLD although lipoprotein X level was low. Acquired LCAT deficiencies are extremely rare with only 7 known cases including ours. Patients with undetectable LCAT activity levels develop nephrotic syndrome that requires PSL treatment; cases whose LCAT activity levels can be determined may also develop nephrotic syndrome, but spontaneously recover. Lipoprotein X may play a role in the development of renal impairment in individuals with FLD. However, the effect might be less significant in individuals with acquired LCAT deficiency.

Particle number analysis of lipoprotein subclasses by gel permeation HPLC in patients with cholesteryl ester transfer protein deficiency.

ObjectiveWe previously reported that patients with cholesteryl ester transfer protein (CETP) deficiency (CETP-D) have a higher prevalence of atherosclerotic cardiovascular disease, in spite of increased HDL-C levels. However, characterization of HDL in CETP-D has not been well described. Therefore, we examined HDL particle number (PN) rather than HDL-C level.Approach and resultsNine patients with CETP-D and 9 normolipidemic subjects were enrolled. We performed gel permeation high-performance liquid chromatography (GP-HPLC) analysis, determined the cholesterol and triglyceride composition of all lipoprotein subclasses, and calculated the PN of each subclass, which consisted of 3 VLDL (large, medium, and small), 4 LDL (large, medium, small, and very small), and 5 HDL (very large, large, medium, small, and very small) subclasses. The PNs of large and medium LDL were significantly lower in CETP-D than that in healthy subjects (0.66- and 0.63-fold decrease, respectively; p<0.001), whereas the PN of very small LDL, which is known to be atherogenic, was significantly higher (1.36-fold increase, p = 0.016). The PNs of very large and large HDL in CETP-D were markedly higher than that in healthy subjects (19.9- and 4.5-fold increase, respectively; p<0.001), whereas the PNs of small and very small HDL, which have more potent anti-atherogenic functions, were significantly lower (0.76- and 0.61-fold decrease, respectively; p<0.001).ConclusionWe have assessed the PNs of detailed subclasses of patients with CETP-D for the first time. The PN of larger HDL was markedly increased, that of smaller HDL was decreased, and that of very small LDL was increased, suggesting that CETP-D has pro-atherogenic lipoprotein properties.

Effect of Desulfonation of Lignosulfonate on the Properties of Poly(Lactic Acid)/Lignin Composites

To utilize the lignin generated by the paper industry and reduce the cost of poly(lactic acid) (PLA), PLA/lignin composites were prepared from PLA and different ratios of lignosulfonate (LS) or desulfonated lignosulfonate (DLS) particles using a casting method. The physicochemical properties of the lignins were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), gel permeation-high performance liquid chromatography (GPC), and elementary analysis. The results indicate that the sulfur content of the original LS was successfully reduced to half by desulfonation to produce DLS, which was found to have a higher thermal stability and a lower average molecular weight than LS. Additionally, the thermal stability, crystallization, compatibility, mechanical, hydrophobicity, and optical properties of the PLA/lignin composites were also meticulously evaluated. Comparison of the PLA/DLS and PLA/LS composites revealed that the incorporation of DLS into PLA improved compatibility, thermal stability (T5% and Tmax), and hydrophobicity, while the mechanical properties remained almost unchanged. In addition, both PLA/DLS and PLA/LS exhibited UV light absorption capacity. Finally, the low-rate addition of both LS (10%) and DLS (5%) accelerated the crystallization of PLA, but crystallization was delayed with higher lignin content.

Cholesterol concentrations in lipoprotein fractions separated by anion-exchange–high-performance liquid chromatography in healthy dogs and dogs with hypercholesterolemia

Anion-exchange (AEX)–high-performance liquid chromatography (HPLC) for measurement of cholesterol can be used to separate serum lipoproteins (high-density lipoprotein (HDL); low-density lipoprotein (LDL); intermediate-density lipoprotein (IDL); very-low-density lipoprotein (VLDL)) in humans. However, AEX–HPLC has not been applied in veterinary practice. We had three objectives: (i) the validation of AEX-HPLC methods including the correlation of serum cholesterol concentration in lipoprotein fraction measured by AEX–HPLC and gel permeation–HPLC (GP–HPLC) in healthy dogs and those with hypercholesterolemia was investigated; (ii) the reference intervals of lipoprotein fractions measured by AEX–HPLC from healthy dogs (n=40) was established; (iii) lipoprotein fractions from the serum of healthy dogs (n=12) and dogs with hypercholesterolemia (n=23) were compared. Analytic reproducibility and precision of AEX–HPLC were acceptable. Positive correlation between serum concentrations of total cholesterol (Total-Chol), HDL cholesterol (HDL-Chol), LDL cholesterol (LDL-Chol)+IDL cholesterol (IDL-Chol), and VLDL cholesterol (VLDL-Chol) was noted for AEX–HPLC and GP–HPLC in healthy dogs and dogs with hypercholesterolemia. Reference intervals measured by AEX–HPLC for serum concentrations of Total-Chol, HDL-Chol, and LDL-Chol were determined to be 2.97–9.32, 2.79–6.57, 0.16–3.28mmol/L (2.5–97.5% interval), respectively. Furthermore, there was significant difference in lipoprotein profiles between healthy and dogs with hypercholesterolemia. These results suggest that AEX–HPLC can be used to evaluate lipoprotein profiles in dogs and could be a new useful indicator of hyperlipidemia in dogs.

Improved Quantitation of Gluten in Wheat Starch for Celiac Disease Patients by Gel-Permeation High-Performance Liquid Chromatography with Fluorescence Detection (GP-HPLC-FLD).

Purified wheat starch (WSt) is commonly used in gluten-free products for celiac disease (CD) patients. It is mostly well-tolerated, but doubts about its safety for CD patients persist. One reason may be that most ELISA kits primarily recognize the alcohol-soluble gliadin fraction of gluten, but insufficiently target the alcohol-insoluble glutenin fraction. To address this problem, a new sensitive method based on the sequential extraction of gliadins, glutenins, and gluten from WSt followed by gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD) was developed. It revealed that considerable amounts of glutenins were present in most WSt. The gluten contents quantitated by GP-HPLC-FLD as sum of gliadins and glutenins were higher than those by R5 ELISA (gluten as gliadin content multiplied by a factor of 2) in 19 out of 26 WSt. Despite its limited selectivity, GP-HPLC-FLD may be applied as confirmatory method to ELISA to quantitate gluten in WSt.

Recent Advances in Analytical Methods on Lipoprotein Subclasses: Calculation of Particle Numbers from Lipid Levels by Gel Permeation HPLC Using “Spherical Particle Model”

Recently, we developed an analytical method for determining the lipid levels and particle numbers in lipoprotein subclasses covering a wide size range from chylomicrons to small high density lipoproteins, by using gel permeation high-performance liquid chromatography (GP-HPLC). The challenges in analytical methods on lipoprotein subclasses have been addressed from 1980 by Hara and Okazaki using commercial TSK gel permeation columns. Later, the improvements in the hardware, separation and detection of lipoproteins, and the data processing software, using a Gaussian distribution approximation to calculate lipid levels of lipoprotein subclasses, have been extensively utilized in these analytical methods for over thirty years. In this review, we describe on the recent advances in analytical methods on lipoprotein subclasses based on various techniques, and the calculation of particle numbers from lipid levels by GPHPLC using the "spherical particle model". Free/ester ratio of cholesterol in particular lipoprotein subclass was accurately estimated from triglyceride, total cholesterol (free and esterified) and the size of the particle based on this model originally proposed by Shen and Kezdy.

Analysis of lipoprotein profiles of healthy cats by gel-permeation high-performance liquid chromatography.

Density gradient ultracentrifugation (DGUC) and gel electrophoresis are conventionally used to obtain lipoprotein profiles of animals. We recently applied high-performance liquid chromatography with a gel permeation column (GP-HPLC) and an on-line dual enzymatic system to dogs for lipoprotein profile analysis. We compared the GP-HPLC with DGUC as a method to obtain a feline lipoprotein profile. The lipoprotein profiles showed large and small peaks, which corresponded to high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively, whereas very low-density lipoprotein (VLDL) and chylomicron (CM) were only marginally detected. This profile was very similar to that of dogs reported previously. Healthy cats also had a small amount of cholesterol-rich particles distinct from the normal LDL or HDL profile. There was no difference in lipoprotein profiles between the sexes, but males had a significantly larger LDL particle size (P=0.015). This study shows the feasibility of GP-HPLC for obtaining accurate lipoprotein profiles with small sample volumes and provides valuable reference data for healthy cats that should facilitate diagnoses.

Recent developments in analytical methods for tracing gluten

According to Codex only foods not exceeding a level of 20 mg gluten/kg may bear a gluten-free label. This also sets the standard for analytical methods for gluten detection. In this paper the currently used methods for gluten analysis are reviewed and new developments are discussed. At the moment, the most commonly used methods are ELISA-based, but also PCR-based methods have been successfully employed. Proteomics-based methods such as reversed-phase (RP-) or gel permeation (GP-) high-performance liquid chromatography (HPLC) have been widely used for characterisation of cereal proteins. Methods combining mass spectrometry and liquid chromatography (LC-MS/MS) are the most promising non-immunological approaches for accurate quantitation of gluten traces. However, due to its requirement of expensive equipment and expertise it is not widely used for routine analysis. New developments include immunosensors, aptamers, microarrays, and multianalyte profiling. Despite the merits and challenges of the different methods, the need for an independent reference method and a generally applicable reference material remain.