Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein.

The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed mutagenesis and expressed in Chinese hamster ovary Flp-In cells. The apparent size of each of the six PLTP mutants was slightly less than that of wild type by Western blot, indicating that all six sites are glycosylated or utilized. The size of the carbohydrate at each N-glycosylation site ranged from 3.14 to 4.2kDa. The effect of site-specific N-glycosylation removal on PLTP secretion varied from a modest enhancement (15% and 60%), or essentially no effect, to a reduction in secretion (8%, 14% and 32%). Removal of N-glycosylation at any one of the six glycosylation sites resulted in a significant 35-78% decrease in PLTP activity, and a significant 29-80% decrease in PLTP specific activity compared to wild type. These data indicate that although no single N-linked carbohydrate chain is a requirement for secretion or activity, the removal of the carbohydrate chains had a quantitative impact on cellular secretion of PLTP and its phospholipid transfer activity.

Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study.

The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) (Lp(a)), Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examination cycle 5 in subjects participating in the Framingham Offspring Study who were free of CHD. After a mean follow-up of 12.3 years, 98 men and 47 women developed new CHD events. In multivariate analysis, the hazard ratio of CHD was approximately two-fold greater in men in the upper tertile of plasma Lp(a) levels, relative to those in the bottom tertile (P < 0.002). The apo(a) isoform size contributed only modestly to the association between Lp(a) and CHD and was not an independent predictor of CHD. In multivariate analysis, Lp(a) cholesterol was not significantly associated with CHD risk in men. In women, no association between Lp(a) and CHD risk was observed. Elevated plasma Lp(a) levels are a significant and independent predictor of CHD risk in men. The assessment of apo(a) isoform size in this cohort does not add significant information about CHD risk. In addition, the cholesterol content in Lp(a) is not a significant predictor of CHD risk.

Apolipoprotein E highly correlates with AbetaPP- and tau-related markers in human cerebrospinal fluid.

We assessed cerebrospinal fluid (CSF) levels of apolipoprotein E (apoE), phospholipid transfer protein (PLTP) activity, cholesterol, secreted amyloid-beta protein precursor alpha and beta (sAbetaPPalpha, sAbetaPPbeta), amyloid-beta peptides 1-40 (Abeta_{40}) and 1-42 (Abeta_{42}), total tau and tau phosphorylated at threonine 181 (pTau) in neurologically healthy, cognitively intact adults. ApoE significantly correlated with sAbetaPPalpha (r = 0.679), sAbetaPPbeta (r = 0.634), Abeta_{40} (r = 0.609), total and pTau (r = 0.589 and r = 0.673, respectively, all p < 0.001), PLTP activity (r = 0.242, p = 0.002) and cholesterol (r = 0.194, p < 0.01). PLTP activity significantly correlated with sAbetaPPalpha (r = 0.292), sAbetaPPbeta (r = 0.281), total and pTau (r = 0.265 and 0.258, respectively; all p

Cerebrospinal fluid apolipoprotein E and phospholipid transfer protein activity are reduced in multiple sclerosis; relationships with the brain MRI and CSF lipid variables.

Apolipoprotein E (apoE), phospholipid transfer protein (PLTP) activity, lipids, total tau and beta amyloid 1-42 (Aß42) were measured in cerebrospinal fluid (CSF) from controls (n=38) and multiple sclerosis (MS) patients (n=91). ApoE and PLTP activity were significantly reduced in MS compared to non-inflammatory disease controls (NINDC; p<0.05). In NINDC and MS, apoE correlated with PLTP activity (rs=0.399 and 0.591, respectively), Aß42 (rs= 0.609 and 0.483, respectively), and total tau (rs=0.748 and 0.380, respectively; all p<0.05). CSF apoE and PLTP significantly contributed to the variance of the normalized brain volume (NBV) and T2 lesion volume in MS (p<0.001 and p<0.05, respectively). ApoE correlated with CSF cholesterol and 24-hydroxycholesterol in all groups; PLTP activity correlated with CSF cholesterol in controls (p<0.05).

Evaluation of a new homogenous method for detection of small dense LDL cholesterol: comparison with the LDL cholesterol profile obtained by density gradient ultracentrifugation.

BACKGROUND: Small, dense LDL (sdLDL) are highly atherogenic, and evidence indicates that sdLDL are useful predictors of cardiovascular disease. We evaluated a homogeneous method for the quantitative determination of sdLDL cholesterol (sdLDL-C) and compared it to the LDL-cholesterol profile obtained by density gradient ultracentrifugation (DGUC). METHODS: sdLDL-C was measured in plasma and in lipoprotein fractions obtained by DGUC using the sdLDL-EX "Seiken" kit. RESULTS: sdLDL-C was only present in dense or intermediate LDL fractions obtained by DGUC. Plasma sdLDL-C levels were inversely corelated to the LDL relative floatation rate. The proportion of LDL-C that is sdLDL-C increases as a function of LDL density from a median of 19% for very buoyant LDL to 91% for very dense LDL particles. Plasma sdLDL-C level was significantly correlated with plasma triglyceride (TG) (n=840, r(s)=0.710, p<0.001). Among subjects with plasma TG>150 mg/dl, 75% had sdLDL-C>30 mg/dl, whereas among those with TG ≤ 150 mg/dl, only 17% had sdLDL-C>30 mg/dl. CONCLUSIONS: This precise and fully automated method for measuring plasma levels of sdLDL-C will allow the analysis of large number of samples in routine laboratories which will facilitate the evaluation of the clinical usefulness of sdLDL as a risk factor for CHD.

Association of Apo(a)isoform size with dyslipoproteinemia in male venous thrombosis patients.

BACKGROUND: Lp(a) is a proatherogenic lipoprotein that may also be prothrombotic. Apo(a) size isoforms have differential effects on fibrinolysis. Whereas Lp(a) concentrations have been linked to venous thromboembolic disease (VTE) risk, apo(a) polymorphisms in VTE have not been studied. METHODS: We used a standardized high resolution agarose gel electrophoresis technique to determine apo(a) isoform size, and a Lp(a) immunoassay insensitive to apo(a) size to measure Lp(a) concentration in 46 men with VTE and 46 age-matched healthy controls. RESULTS: Apo(a) isoform distribution in VTE cases and controls was bimodal and VTE patients tended to have more medium-sized isoforms K(4)-(19-27) (54.3% vs. 34.8%, p=0.06). Cases and controls had the same median predominant apo(a) size isoform (23.5 K(4) repeats) and comparable Lp(a) concentrations. However, subgroup analysis based on apo(a) isoform size (K(4)< or =23 or K(4)> or =24) revealed that cases in the K(4)> or =24 subgroup had higher Lp(a) concentrations than the controls in this isofrom subgroup (14.5 mmol vs. 6.6 mmol, p=0.029). Also, dyslipoproteinemia (smaller LDL and HDL particles, higher LDL and lower HDL parameters) was strongly associated with VTE only in this larger apo(a) isoform group. CONCLUSIONS: These observations provide the first evidence that determination of apo(a) isoforms may provide useful novel insights into VTE risk.

Reduced CSF PLTP activity in Alzheimer's disease and other neurologic diseases; PLTP induces ApoE secretion in primary human astrocytes in vitro.

Phospholipid transfer protein (PLTP) plays a pivotal role in cellular lipid efflux and modulation of lipoprotein metabolism. PLTP is distributed widely in the central nervous system (CNS), is synthesized by glia and neurons, and is active in cerebrospinal fluid (CSF). The aims of this study were to test the hypothesis that patients with Alzheimer's disease (AD) have altered PLTP-mediated phospholipid transfer activity in CSF, and to examine the potential relationship between PLTP activity and apolipoprotein E (apoE) levels in CSF. We assessed PLTP activity and apoE concentration in CSF of patients with probable AD (n = 50), multiple sclerosis (MS; n = 9), other neurologic diseases (n = 21), and neurologically healthy controls (n = 40). PLTP activity in AD was reduced compared to that in controls (P < 0.001), with approximately half of the AD patients with PLTP activity values below all controls. Patients with MS had lower PLTP activity than AD patients (P < 0.001). PLTP activity was highly correlated with PLTP mass, as estimated by Western blot (r = 0.006; P < 0.01). CSF PLTP activity positively correlated with apoE concentration in AD (R = 0.435; P = 0.002) and controls (R = 0.456; P = 0.003). Anti-apoE immunoaffinity chromatography and Western blot analyses indicated that some CSF PLTP is associated with apoE-containing lipoproteins. Exogenous addition of recombinant PLTP to primary human astrocytes significantly increased apoE secretion to the conditioned medium. The findings of reduced PLTP activity in AD CSF, and the observation that PLTP can influence apoE secretion in astrocytes suggest a potential link between alterations in the brain lipid metabolism and AD pathogenesis.

Selective and independent associations of phospholipid transfer protein and hepatic lipase with the LDL subfraction distribution.

Phospholipid transfer protein (PLTP), hepatic lipase (HL), and lipoprotein lipase (LPL) have all been reported to be intricately involved in HDL metabolism but the effect of PLTP on the apolipoprotein B-containing lipoproteins relative to that of HL and LPL has not been established. Due to our previous observation of a positive correlation of PLTP activity with plasma apoB and LDL cholesterol, the relationship of PLTP with the LDL subfractions was investigated and compared with that of HL and LPL. Plasma lipoproteins from 50 premenopausal women were fractionated by density gradient ultracentrifugation. Correlations were calculated between the cholesterol concentration of each fraction and plasma PLTP, HL, and LPL activity. Plasma PLTP activity was highly, positively, and selectively correlated with the cholesterol concentration of the buoyant LDL/dense IDL fractions, yet demonstrated a complete absence of an association with the dense LDL fractions. In contrast, HL was positively correlated with the dense LDL fractions but showed no association with buoyant LDL. LPL was also positively correlated with several buoyant LDL fractions; however, the correlations were weaker than those of PLTP. PLTP and LPL were positively correlated and HL was negatively correlated with HDL fractions. The results suggest that PLTP and HL may be important and independent determinants of the LDL subpopulation density distributions.

Differences in reactivity of antibodies to active versus inactive PLTP significantly impacts PLTP measurement.

Due to conflicting reports concerning the relationship between phospholipid transfer protein (PLTP) activity and mass in plasma, the protein concentration and activity of PLTP were assessed in fractions isolated by fast protein liquid chromatography from the plasma of healthy normolipidemic individuals. Using both polyclonal and monoclonal antibodies, PLTP was identified by Western blot analysis after both SDS and non-denaturing gradient gel electrophoresis, and quantitated by dot blot. PLTP activity was determined using a labeled vesicle/HDL assay. PLTP mass corresponded substantially with the activity distribution using the polyclonal antibody on dot blot with some inactive PLTP being present. However, the monoclonal antibody preferentially reacted with inactive PLTP, primarily associated with LDL and large HDL, overestimating inactive PLTP. Western blot analysis of non-denaturing gradient gels, using the polyclonal antibody, indicated that active PLTP was associated with numerous discrete HDL subpopulations (7.6-12.0 nm) with the major portion being 9-12 nm. Inactive PLTP was associated with particles of 12 to >17 nm. The monoclonal antibody demonstrated a different pattern of reactivity on gradient gels, showing strong reactivity with the inactive PLTP in particles of 12 to >17 nm, but less reactivity with particles of 7.6-12 nm. The differences in reactivities of antibodies for active versus inactive PLTP can account for some of the discrepancies reported in the literature regarding the relationship between PLTP mass and activity.

Proyecto de estandarización para la medición de apolipoproteínas A-I y B: II evaluación y selección de candidatos a materiales de referencia / Proyect of standarization for the measurement of apolipoproteins A-I y B: evaluation and selection of candidates by material of reference

La primera fase de un estudio internacional en colaboración para estandarizar sistemas de análisis para la medición de apo A-I y B, demostró que la uniformidad de tales mediciones puede conseguirse si se usan materiales de referencia comunes, adecuados para calibrar los distintos sistemas. Durante esta fase, varios candidatos a materiales de referencia fueron seleccionados para una posterior evaluación como materiales de referencia internacionales. El objetivo de la segunda fase del estudio, fue evaluar la linealidad y el paralelismo o proporcionalidad de los candidatos a materiales seleccionados en la fase 1, y determinar si alguno de ellos puede proponerse como material de referencia internacional. Para evaluar los materiales de referencia propuestos, fueron usados 37 sistemas de análisis, para apo A-I y 38 para apo B, abarcando a 23 fabricantes y 5 laboratorios de investigación. Para apo A-I fueron propuestas 2 preparaciones liofilizadas, SP1 de Behringwerke AG, Alemania, y SP2 de Daiichi Pure Chemicals Co, Japón; y para apo B, se propusieron 2 preparaciones líquidas, SP3 de Behringwerke AG, Alemania y SP4 de Reagents Applications, USA. La linealidad de los candidatos a materiales de referencia fue comparada con la de un pool de sueros frescos congelados, o materiales de referencia sérico provisorio (MRSP), distribuido a todos los participantes y con la de un pool de suero fresco (PSF), preparado por cada participante. El C.V. total entre laboratorios, después de una calibración uniforme, medido sobre 6 muestras de sueros normolipémicos, fue cerca del 6%para apo A-I y alrededor del 7%para apo B. Los candidatos a materiales de referencia SP1 y SP3 exhibieron linealidad y paralelismo similar al del pool de suero fresco congelado y tenían CVs interlaboratorio menores o similares a los obtenidos en muestras de suero normolipémicos. Por lo tanto, estos dos materiales fueron seleccionados como candidatos internacionales a materiales de referencia