Polyacrylamide Gradient Gel Electrophoresis Research Articles

Heritability of LDL peak particle diameter in the Quebec Family Study.

LDL size has been associated with the risk of coronary heart disease. The objective of the present study was to verify whether familial factors influence LDL peak particle diameter (LDL-PPD), a quantitative trait reflecting the size of the major LDL subclass. LDL-PPD was measured by 2-16% polyacrylamide gradient gel electrophoresis in 681 members of 236 nuclear families participating in the Quebec Family Study. LDL-PPD was adjusted for age (LDL-PPD1), age and body mass index (LDL-PPD2), or age, body mass index, and plasma triglyceride levels (LDL-PPD3) separately in men and women. The residual scores were used to test for familial aggregation, using an ANOVA and to compute maximum likelihood estimates of familial correlations. The ANOVA test revealed that family lines accounted for 47.4%, 46.7%, and 48.9% of the variance in the LDL-PPD1, LDL-PPD2, and LDL-PPD3 phenotypes, respectively. The pattern of familial correlations revealed no significant spouse correlations but significant parent-offspring and sibling correlations for the three LDL-PPD phenotypes, with maximal heritability estimates of 59%, 58%, and 52% for LDL-PPD1, LDL-PPD2, and LDL-PPD3, respectively. These results suggest that LDL-PPD strongly aggregates in families, and that the familial resemblance appears to be primarily attributable to genetic factors. Genes responsible for this genetic contribution remain to be identified.

Allophycocyanin complexes from the phycobilisome of a thermophilic blue-green alga Myxosarcina concinna Printz

The core polypeptide components of the intact phycobilisomes (PBSs) prepared by the sucrose gradients in 0.9 M phosphate buffer from a thermophilic cyanobacterium Myxosarcina concinna Printz were investigated. Three allophycocyanins, designated AP1, AP2, and AP3, of the PBS cores were successfully prepared by using the gradient polyacrylamide gel electrophoresis (PAGE) performed in neutral, instead of alkaline, buffer system. The spectral properties of AP2 and AP3 demonstrated that they both had fluorescence emission maxima at 684/685 nm at 77 K, which was identical to those of the intact PBSs, and showed the absorption of allophycocyanin B (AP-B) subunit. Sodium dodecyl sulfate–PAGE revealed that the three biliprotein complexes were all composed of heterogeneous subunits and two more linker polypeptides (Ls), AP1 α 22.3α 19.5β 17.4β 15.7L 13.8L 11.3L 9.5, AP2 α 22.3α 19.5β 17.4β 15.7β 15.1L 11.3L 9.5, and AP3 α 22.3α 19.5β 17.4β 15.7β 15.1L 11.3L 9.5L 8.3. Compared with the characteristics of AP1, β 15.1, which belonged to the β subunit group, was the AP-B subunit of AP2 and AP3. Because AP2 was only obtained together with the PBS by the aid of 2% (v/v) Triton X-100, but not AP3, it was closely related to anchoring the PBS core on thylakoid membranes though the polypeptide analysis showed that AP2 had no core-membrane linker (L CM). Aggregates of the three AP biliproteins were proposed based on the present results, and their functions in the PBS core construction and the energy transfer to PS II and PS I were discussed.

Sulfite and base for the treatment of familial amyloidotic polyneuropathy: two additive approaches to stabilize the conformation of human amyloidogenic transthyretin.

Recently, we presented evidence that sulfite protects transthyretin (TTR) from normal human individuals and heterozygotes with amyloidogenic TTR mutations against the decay of tetramers into monomers. In this paper we demonstrate a stabilizing effect of sulfite on TTR tetramers from a familial amyloidotic polyneuropathy (FAP) patient homozygous for the most-common amyloidogenic TTR-V30 M mutation. We compare the conformational stability of partially sulfonated TTR from a heterozygote for normal TTR and amyloidogenic TTR-V30 M with the stability of untreated TTR from a compound heterozygote for amyloidogenic TTR-V30 M and TTR-T119 M known to have only minor or no problems of FAP. Using a combination of polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) gradient PAGE we demonstrate that TTR dimers containing amyloidogenic TTR mutations decay into monomers at pH<7.4. Increasing the pH by some 0.2 units within physiological ranges, i.e., pH 7.0-7.4, and sulfonation of TTR were observed to have additive inhibitory effects on the transition of dimers into monomers. We conclude that mild acidifying episodes in the interstitial volume of tissues at risk for amyloidosis could contribute to the development of FAP. Early and permanent efforts to counteract acidosis by treatment with base could possibly help to delay the onset of the disease. The intake of sulfite could support these efforts.

Effect of different forms of dietary hydrogenated fats on LDL particle size

Dietary trans fatty acids (FAs), which are formed during the process of hydrogenating vegetable oil, are known to increase plasma LDL-cholesterol concentrations. However, their effect on LDL particle size has yet to be investigated. We investigated the effect of trans FA consumption on the electrophoretic characteristics of LDL particles. Eighteen women and 18 men each consumed 5 experimental diets in random order for 35-d periods. Fat represented 30% of total energy intake in each diet, with two-thirds of the fat in the form of semiliquid margarine (0.6 g trans FAs/100 g fat), soft margarine (9.4 g trans FAs/100 g fat), shortening (13.6 g trans FAs/100 g fat), stick margarine (26.1 g trans FAs/100 g fat), or butter, which was low in trans FAs (2.6 g trans FAs/100 g fat) but rich in saturated fat. LDL particle size and distribution were characterized by nondenaturing, 2-16% polyacrylamide gradient gel electrophoresis. Relative to the LDL particle size observed after consumption of the butter-enriched diet, LDL particle size decreased significantly and in a dose-dependent fashion with increasing amounts of dietary trans FAs (P < 0.001). Cholesterol concentrations in large (> 260 A) and medium-sized (255-260 A) LDL particles also increased proportionately to the amount of trans FAs in the diet. Consumption of dietary trans FAs is associated with a deleterious increase in small, dense LDL, which further reinforces the importance of promoting diets low in trans FAs to favorably affect the lipoprotein profile.

Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin.

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.

Platelet-activating factor acetylhydrolase is mainly associated with electronegative low-density lipoprotein subfraction.

Electronegative LDL [LDL(-)], a modified subfraction of LDL present in plasma, induces the release of interleukin-8 and monocyte chemotactic protein-1 from cultured endothelial cells. We demonstrate that platelet-activating factor acetylhydrolase (PAF-AH) is mainly associated with LDL(-). LDL(-) had 5-fold higher PAF-AH activity than the nonelectronegative LDL subfraction [LDL(+)] in both normolipemic and familial hypercholesterolemic subjects. Western blot analysis after SDS-PAGE confirmed these results, because a single band of 44 kDa corresponding to PAF-AH appeared in LDL(-) but not in LDL(+). Nondenaturing polyacrylamide gradient gel electrophoresis demonstrated that PAF-AH was bound to LDL(-) regardless of LDL size. In accordance with the above findings, nonesterified fatty acids, a cleavage product of PAF-AH, were increased in LDL(-) compared with LDL(+). The high PAF-AH activity observed in LDL(-) could be related to the proinflammatory activity of these lipoproteins toward cultured endothelial cells.

Effect of obesity on HDL and LDL particle sizes in carriers of the null P207L or defective D9N mutation in the lipoprotein lipase gene: the Québec LipD Study.

We have recently demonstrated that French Canadians bearing a mutation in the lipoprotein lipase (LPL) gene present an impaired lipoprotein-lipid profile characterized by small low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles compared with healthy subjects. It has also been documented that obesity has a significant impact on HDL and LDL particle sizes. To examine the extent to which obesity modulates HDL and LDL particle sizes among carriers of mutations in the LPL gene. Analyses were carried out in 206 heterozygous carriers of the D9N mutation (N=118) or the P207L mutation (N=88). Lipoprotein particle sizes were measured on whole plasma by nondenaturing polyacrylamide gradient gel electrophoresis. In general, body mass index (BMI) and waist circumference were significant correlates of LDL and HDL particle sizes among heterozygous carriers of the P207L or D9N mutation in the LPL gene, with relatively similar associations among men and women. Multivariate analyses indicated that variations in waist circumference but not BMI were an independent predictor of variations in both HDL particle size (5.2%, P=0.0005) and LDL particle size (5.9%, P=0.01) in the entire group of heterozygotes for LPL mutation in a model that included the nature of the LPL mutation (D9N vs P207L), gender, age, cholesterol and plasma TG levels. Interestingly, there was a significant interaction between plasma TG levels and waist circumference or BMI in modulating HDL particle size. Indeed, an increased waist circumference or BMI was associated with a significant reduction in HDL particle size among subjects with plasma TG levels <or=3.5 mmol/l, but not among those with marked hypertriglyceridemia (TG levels >3.5 mmol/l). These results suggest that abdominal obesity, more so that overall obesity, is an important determinant of variations in LDL and HDL particle size among heterozygous carriers of mutations in the LPL gene, perhaps further contributing to modulate the risk of CHD in these individuals.

Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase

Although previous chemical modification studies have suggested several residues to be involved in the maintenance of the quaternary structure of glutamate dehydrogenase (GDH), there are conflicting views for the polymerization process and no clear evidence has been reported yet. In the present study, cassette mutagenesis at seven putative positions (Lys333, Lys337, Lys344, Lys346, Ser445, Gly446, and His454) was performed using a synthetic human GDH gene to examine the polymerization process. Of the mutations at the seven different sites, only the mutagenesis at His454 results in depolymerization of the hexameric GDH into active trimers as determined by HPLC gel filtration analysis and native gradient polyacrylamide gel electrophoresis. The mutagenesis at His454 has no effects on expression or stability of the protein. The K M values for NADH and 2-oxoglutarate were 1.5-fold and 2.5-fold greater, respectively, for the mutant GDH than for wild-type GDH, indicating that substitution at position 454 had appreciable effects on the affinity of the enzyme for both NADH and 2-oxoglutarate. The V max values were similar for wild-type and mutant GDH. The k cat/ K M value of the mutant GDH was reduced up to 2.8-fold. The decreased efficiency of the mutant, therefore, results from the increase in K M values for NADH and 2-oxoglutarate. The results with cassette mutagenesis and HPLC gel filtration analysis suggest that His454 is involved in the polymerization process of human GDH.

Comparison of Low-Density Lipoprotein Size by Polyacrylamide Tube Gel Electrophoresis and Polyacrylamide Gradient Gel Electrophoresis

We evaluated a low-density lipoprotein (LDL) subfraction separation method using polyacrylamide tube gel electrophoresis (PTGE) and compared it with the reference method, polyacrylamide gradient gel electrophoresis (PGGE-REF). Excellent intra-assay and interassay coefficients of variation were obtained (<4%) for PTGE. For 102 subjects, LDL subclasses correlated most significantly with triglyceride (TG) level, high-density lipoprotein (HDL) cholesterol level, total cholesterol/HDL cholesterol ratio, and non-HDL cholesterol level (P < .05). The distribution of large LDL (76%) was predominant for subjects with low TG levels (<150 mg/dL [1.69 mmol/L]), while distribution of small LDL (79%) was predominant for subjects with high TG levels (>200 mg/dL [2.26 mmol/L]). Excellent agreement between the methods was observed (weighted kappa = 0.78). Of 51 samples classified as small, dense LDL by PGGE-REF, none were misclassified as large LDL and 4 as intermediate LDL by PTGE (92% concordance); of 44 samples classified as large LDL by PGGE-REF, 3 were misclassified as small and 7 as intermediate by PTGE (77% concordance). The PTGE method is precise and compares favorably with PGGE-REF. It has the advantage of being simple, less expensive, and more suitable for use in the clinical laboratory.

Effect of Plasma C-Reactive Protein Levels in Modulating the Risk of Coronary Heart Disease AssociatedWith Small, Dense, Low-Density Lipoproteins in Men(The Quebec Cardiovascular Study)

This purpose of this study was to investigate how plasma C-reactive protein (CRP), a nonspecific acute-phase reactant, modulates the risk of coronary heart disease (CHD) associated with the small, dense, low-density lipoprotein (LDL) phenotype. LDL particle size and plasma CRP were measured in the Quebec Cardiovascular Study cohort of 2,025 men free of CHD at baseline, among whom 103 had a first CHD event during a 5-year follow-up period. Plasma CRP levels were measured using the Behring Latex-Enhanced (highly sensitive) CRP assay. LDL particle size phenotype was characterized using 2% to 16% polyacrylamide gradient gel electrophoresis. There were weak but significant associations between plasma CRP levels and features of LDL size, such as the proportion of LDL with a diameter <255 Å (r = 0.09, p <0.001) and LDL peak particle size (r = −0.09, p <0.001). Variations in plasma CRP levels modulated the risk of CHD associated with small LDL peak particle size (relative risk 4.3 vs 2.5 in men with high vs low plasma CRP levels, respectively) and with an elevated proportion of LDL <255 Å (relative risk 6.6 vs 3.0). Thus, increased plasma CRP levels further elevate the risk of CHD associated with having small, dense LDL particles.

Influence of common variants in the CETP, LPL, HL and APO E genes on LDL heterogeneity in healthy, middle-aged men

Low density lipoprotein (LDL) particle size is a genetically influenced trait associated with coronary heart disease (CHD). This study investigates the effects of genetic variation in plasma factors with important roles in lipoprotein metabolism on LDL heterogeneity. Common variants in the cholesteryl ester transfer protein (CETP-629C/A), lipoprotein lipase (LPL S447X), hepatic lipase (HL-480C/T) and apolipoprotein E (apoE e2/e3/e4) genes were studied in relation to LDL particle size distribution in 377 healthy, middle-aged men. A high-resolution polyacrylamide gradient gel electrophoresis technique was used to measure plasma concentrations of four LDL subfractions. The CETP-629A and LPL 447X alleles were associated with moderately increased LDL peak particle size. In contrast, the apoE e4 allele was associated with a marked reduction in LDL peak particle size and an increased relative proportion and plasma concentration of small, dense LDL. An interaction between the HL-480C/T and apo E polymorphisms contributed significantly to increased plasma concentration of small, dense LDL (LDL-III) in HL-480T carriers. In summary, the investigated polymorphisms were associated with diverse effects on the LDL particle size distribution, consistent with respect to protein function and proposed association with CHD risk. The observed associations were further modulated by gene–gene and gene–environment interactions.

Mycobacterium tuberculosis Hsp16.3 Nonamers are Assembled and Re-assembled via Trimer and Hexamer Intermediates

Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis proposed to form specific trimer-of-trimers structures, acts as a molecular chaperone in vitro. The assembly and re-assembly mechanisms of this oligomeric protein were studied and compared using in vitro transcription/translation and denaturization/renaturization systems. Analysis using a combination of non-denaturing pore gradient polyacrylamide gel electrophoresis, chemical cross-linking, and size-exclusion chromatography demonstrate that the predominant form of Hsp16.3 produced in the in vitro transcription/translation system is the trimer, which can be further assembled into a nonameric structure via a hexamer intermediate in the presence of purified exogenous Hsp16.3 proteins. Meanwhile, an “inert” Hsp16.3 dimer, which does not seem to participate in nonamer assembly but may be involved in forming other forms of Hsp16.3, was also detected in the in vitro expression system. On the other hand, our current data clearly show that the re-assembly of Hsp16.3 nonamers also occurs via a very similar mechanism, with the formation of trimers and hexamers. The presence of high levels of macromolecular crowding protein agent in the in vitro expression system promoted the formation of the nonamers to a very limited degree, indicating that the assembly of proteins like Hsp16.3 may depend mainly on its own concentration instead of those of the macromolecules in the environment.

Comparison of effectiveness of statin monotherapy versus statin and niacin combination therapy in primary prevention and effects on calcified plaque burden

T combination of a statin and niacin is increasingly being prescribed for treatment of combined lipid disorders, and has recently been shown to produce a dramatic decrease in clinical events and atherosclerosis progression, measured by quantitative coronary angiography in secondary prevention patients.1 This study was designed to determine whether calcified plaque progression, determined by electron beam tomography (EBT), differs in groups of primary prevention patients treated with statins alone compared with statin and niacin combination therapy. In addition, the correlations between the change in calcified plaque burden and changes in low-density lipoprotein (LDL) particle size and subgroups were evaluated. • • • One hundred sixty-two consecutive asymptomatic patients with EBT evidence for subclinical atherosclerosis, who were treated with a statin alone or in combination with niacin (Niaspan; Kos Pharmaceuticals, Miami, Florida) and underwent repeat EBT evaluation, comprised the patient population. This was an observational study; serial EBT evaluation and lipidlowering agents and doses employed were chosen by the treating physicians based on clinical considerations, and not according to criteria set by the investigators. All patients gave consent for their data to be used in subsequent analyses. No patient had a history of clinical coronary artery disease. Hypertension was defined as the current or recommended use of antihypertensive medications for blood pressure control. Smoking was defined as current tobacco usage, and diabetes was defined as the current or recommended use of diet or medication to decrease blood sugar. Family history of coronary artery disease was defined as established disease in first-degree male relatives 56 years of age or female relatives 66 years of age. Body mass index was calculated as weight (kilograms)/height (meters squared). Lipid measurements were obtained at the time of both EBT evaluations. EBT was performed using an Imatron C-150 scanner (GE/Imatron, South San Francisco, California) and previously described acquisition protocol.2 The coronary artery calcium score was calculated with the Agatston method.3 The calcium volume score, a parameter of plaque burden that is density independent, was calculated according to the method of Callister et al.4 The calcium percentile, an age and gender corrected index of plaque prematurity, was derived from the University of Illinois asymptomatic patient database. EBT studies done before and after treatment were reviewed side-by-side for comparability of analysis by an experienced observer (HSH), blinded to the treatment effects. Changes in coronary calcium and calcium volume scores were calculated on an absolute basis and as a percent change per year. To avoid skewing the data by large percentage changes in a few patients with lower scores, the percent change per year for the treated and untreated groups was calculated by dividing the mean annualized change by the mean baseline values for the groups as a whole, rather than by the mean of the percent change per year for each patient. Fasting plasma lipid and lipoprotein cholesterol concentrations were determined by the methods of the Lipid Research Clinics in all patients.5 In 53 patients treated with niacin, densitometric measurements of LDL subspecies were carried out using custom made, triple gradient (S3), 2% to 16% polyacrylamide gradient gel electrophoresis (S3GGE) of plasma samples (Berkeley HeartLab, Burlingame, California).6 LDL peak particle diameter was measured and LDL subclass distribution was determined in 3 large regions (I, IIa, IIb) and 4 small regions (IIIa, IIIb, IVa, IVb). LDL subclass pattern was classified based on LDL peak particle diameter, number and position of peaks, and From the Beth Israel Medical Center, New York, New York; and Kronos Longevity Research Institute, Phoenix, Arizona. Dr. Hecht’s address is: PO Box 450, 9 Woodland Road, Brookside, New Jersey 07926. E-mail: hhecht@aol.com. Manuscript received July 22, 2002; revised manuscript received and accepted September 20, 2002. TABLE 1 Patient Characteristics

Non-denaturing polyacrylamide gradient gel electrophoresis for the diagnosis of dysbetalipoproteinemia

Dysbetalipoproteinemia, an uncommon but highly atherogenic mixed hyperlipidemia due to the accumulation of remnants of triglyceride-rich lipoproteins, is characterized by cholesterol-enriched VLDL that migrates in the beta-position on agarose gels. The demonstration of a broad beta-band on agarose gel electrophoresis of plasma is an insensitive method and ultracentrifugation is an impractical method of diagnosing this condition. Non-denaturing polyacrylamide gradient gel electrophoresis (PGGE) was investigated as a screening method for the diagnosis of dysbetalipoproteinemia. A minigel procedure separating the Sudan Black prestained apolipoprotein B (apoB)-containing lipoproteins on a 2-8% polyacrylamide gel at 4 degrees C overnight was analyzed for ultracentrifugally and genetically proven dysbetalipoproteinemic subjects as well as matched controls for mixed hyperlipidemia. Visual inspection revealed that the presence of only small VLDL- and IDL-like particles in untreated patients was highly sensitive (72%) and specific (95%) for dysbetalipoproteinemia. Videodensitometric analysis of area under the curve for large and small VLDL, as well as IDL and LDL, permitted even better discrimination in subjects whose profiles included some staining in the LDL-like region. A ratio of area under the curve of more than 0.5 for IDL-LDL allowed for a specificity of 100% and a sensitivity of 89% for the diagnosis of dysbetalipoproteinemia. This modified PGGE system may be useful in screening for dysbetalipoproteinemia.

Alpha-crystallin promotes assembly of a trimeric form of Mycobacterium tuberculosis Hsp16.3 in a cell free system.

Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis proposed to form specific trimer-of-trimers structures, acts as a molecular chaperone in vitro. The assembly mechanisms of this oligomeric protein were studied using in vitro transcription/translation systems. Analysis using a combination of non-denaturing pore gradient polyacrylamide gel electrophoresis and size-exclusion chromatography demonstrates that the predominant form of Hsp16.3 produced in the in vitro transcription/translation system is the trimer. Our result indicated that alpha-crystallin (molecular chaperone) remarkably promotes the trimer assembly of Hsp16.3, but does not convert the trimeric form to nonameric form. An "inert" Hsp16.3 dimer, which does not seem to participate in trimer assembly but may be involved in forming other forms of Hsp16.3, was also detected in the in vitro expression system. A latent phase of ~10 min in the appearance of the first detectable species indicated that Hsp16.3 assembly did not occur co-translationally.

LDL-cholesterol/apolipoprotein B ratio is a good predictor of LDL phenotype B in type 2 diabetes.

LDL phenotype B is a component of diabetic dyslipidaemia, but its diagnosis is cumbersome. Our aim was to find easily available markers of phenotype B in a group of type 2 diabetic subjects. We studied 123 type 2 diabetic patients (67.5% male, aged 59.3+/-10.1 years, mean HbA1c 7.4%). Clinical features and fasting total cholesterol, triglyceride, HDL cholesterol, LDL cholesterol (LDLc, using Friedewald's equation and an alternative formula), apolipoprotein B (apoB), lipoprotein (a) and LDL particle size (on gradient polyacrylamide gel electrophoresis) were assessed. Patients with phenotypes A (predominant LDL size > or =25.5 nm) and B (<25.5 nm) were compared, and regression analysis was performed to find the best markers of LDL particle. Cut-off points were obtained and evaluated as predictors of phenotype B (kappa index). Patients with phenotype B (36%) showed higher total cholesterol, triglyceride and apolipoprotein B, and lower HDL cholesterol and LDLc/apoB ratio. Triglyceride was the best predictor of LDL particle size (r=-0.632, p<0.01), but an LDLc/apoB ratio below 1.297 mmol/g detected phenotype B best (sensitivity 65.9%, specificity 92.4%, kappa=0.611). Although triglyceride concentration is the best predictor of LDL size in type 2 diabetes, LDLcholesterol/apolipoproteinB ratio is the best tool to detect phenotype B.

Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site

Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (Glc pA) and idopyranosyluronic acid (Ido pA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (Glc pNAc) residue was lost on workup. The Glc pA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6- O-sulfo glycopyranosylamine (Glc pNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in Glc pNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained Glc pNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.

Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis.

Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.

Determinants of HDL particle size in patients with the null (P207L) or defective (D9N) mutation in the lipoprotein lipase gene: the Québec LipD Study

The aim of the present study was to examine the impact of the defective D9N and the null P207L mutations in the lipoprotein lipase (LPL) gene on high density lipoprotein (HDL) particle size in relation to specific environmental factors such as obesity, gender and menopausal status. Analyses were carried out in 118 heterozygous carriers of the D9N mutation and 88 heterozygous for the P207L mutation. HDL particle size was measured on whole plasma by non-denaturing 4–30% polyacrylamide gradient gel electrophoresis. Although carriers of the P207L mutation presented a more deteriorated lipoprotein–lipid profile compared with carriers of the D9N mutation, there was no difference in HDL particle size between the P207L and D9N carriers (81.9±4.5 vs. 82.7±4.4 Å, respectively, P=0.2). Multivariate analyses indicated that waist circumference ( P=0.001) and HDL cholesterol levels ( P<0.001) were independent predictors of HDL particle size among carriers of the defective D9N mutation. On the other hand, gender ( P=0.03), plasma cholesterol ( P=0.01) and TG ( P=0.04) levels were significant predictors of HDL particle size among carriers of the null P207L mutation in multivariate analyses. These results suggest that the nature of the mutation in the LPL gene modifies the relationship of HDL particle size to other metabolic variables and secondary factors such as abdominal obesity and gender.

HDL particle size: a marker of the gender difference in the metabolic risk profile

A low plasma HDL-cholesterol concentration is an important risk factor for coronary heart disease (CHD) and is often accompanied by increased triglyceride concentrations. Women have generally higher HDL-cholesterol and lower triglyceride concentrations and concomitantly are at lower risk of CHD than men. As HDL particle size is a new and potentially important marker of CHD risk, we have examined the potential gender difference in HDL particle size, assessed by nondenaturing 4–30% polyacrylamide gradient gel electrophoresis, in a sample of men ( n=231) and women ( n=183). Overall, men were characterized by a less favorable lipoprotein–lipid profile, which was accompanied by smaller HDL particle size compared to women. However, when men and women were matched for HDL particle size and compared for their metabolic profile, it was found that both genders were characterized by similar plasma lipoprotein–lipid profile despite the fact that women were characterized by higher levels of total body fat but lower waist girth than men. In summary, HDL particle size is a strong marker of the gender-related difference in the determination of the metabolic risk profile.