Biomedical subjects

J S Parks

Publications and source records attributed to J S Parks.

At least 19 recordsLinked to original sources

PubMed1997

Cloning and in vitro expression of rat lecithin:cholesterol acyltransferase.

Rat lecithin:cholesterol acyltransferase (LCAT) cDNA was obtained by reverse transcriptase/polymerase chain reaction amplification of rat liver total RNA. A consensus sequence was derived from four independent clones from two strains of rats. In vitro expression of rat LCAT cDNA in COS cells resulted in secreted enzyme protein with the same fatty acyl specificity for phospholipase A2 activity and cholesterol esterification as rat plasma LCAT, but different from that of recombinant or human plasma LCAT.

Animals↗

PubMed1997

Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity.

Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98-99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220-241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100:5:1 molar ratio of L-alpha-dimyristoylphosphatidylcholine:cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 A on nondenaturing 4-30% polyacrylamide gel electrophoresis. L-alpha-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent Vmax/apparent Km as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wild-type apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.

Amino Acid Sequence↗

PubMed1997

Amino acid residue 149 of lecithin:cholesterol acyltransferase determines phospholipase A2 and transacylase fatty acyl specificity.

Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleoyl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmitoyl-2-arachidonoyl PC (PAPC). Six recombinant human LCAT cDNA clones were constructed with unique clusters of rat sequence substitutions in the human background spanning the region encoding amino acids 121-296. Media from transfected COS cells expressing each of the constructs were assayed for LCAT cholesterol esterification (CE) or phospholipase A2 (PLA2) activity using substrate particles containing POPC or PAPC. The PAPC/POPC CE activity ratio of the cluster 1 construct (amino acids 149-158) was 1.3, resembling rat LCAT, whereas cluster 2-5 clones produced CE activity ratios <0.3, unchanged from human LCAT. The cluster 6 clone (Y292H/W294F) had an intermediate ratio (0.6). Similar results were observed for LCAT PLA2 activity. In additional studies, position 149 of human LCAT was changed to the rat sequence (hE149A) and compared to a triple mutation containing the remainder of the cluster 1 changes (G151R/E154D/R158Q). CE and PLA2 activity ratio for the hE149A construct was >1.7, similar to rat LCAT, whereas the triple mutation construct retained a ratio similar to human LCAT (<0.6). Thus, a single amino acid substitution (E149A) was sufficient to alter the fatty acyl specificity of human LCAT to that of rat LCAT, with an increase in activity toward PAPC. This is the first example of a point mutation in an enzyme with PLA2 activity that results in an increase in activity toward arachidonic acid.

Alanine↗

PubMed1997

The spectrum of growth-hormone insensitivity.

OBJECTIVES: To summarize the steps that led to the recognition of the Laron syndrome of growth-hormone (GH) insensitivity as a recessive disorder that is caused by mutations in the growth hormone receptor (GHR) gene, to discuss the different types of mutations that have been found in the GHR gene, and to examine whether the degree of growth impairment in affected homozygotes depends on the specific type of GHR mutation. RESULTS: A broad spectrum of abnormalities in the GHR gene have been reported. These abnormalities range from deletions of multiple exons, through deletions of a small number of base pairs, nonsense mutations, missense mutations that alter GH-binding affinity or impair receptor processing, to mutations that exert their deleterious effects by altering messenger RNA splicing and result in the loss of portions of the GHR. CONCLUSIONS: Different abnormalities in the GHR gene have different effects on the concentrations of circulating GH-binding protein that represents the extracellular portion of the GHR. Homozygosity or compound heterozygosity for the different mutations produces a fairly uniform phenotype of severe postnatal growth retardation. The differences in height standard deviation scores between persons appear to depend more on age, sex, and nutritional status than on the specific type of GHR mutation.

Age Factors↗

PubMed1997

Genes regulating hypothalamic and pituitary development.

Several pituitary transcription factors have been identified in the last 3 years. They offer new insights into the processes that direct organogenesis, cell commitment, proliferation and differentiated function. All are DNA-binding proteins, but they have ties to different families of homeodomain proteins. They differ in their distribution and in the timing of their appearance and extinction. The Rathke's pouch homeobox protein (Rpx) has a paired-like homeodomain. In mice, it appears on embryonic day 8.5 (day e8.5) and is gone by day e14.5. Its targets for activation are unknown. Pituitary OTX has a tryptophan--phenylalanine--lysine motif in its homeodomain. It appears early and persists. It shows independent activation of the alpha-glycoprotein subunit (alpha-GSU) and pro-opiomelanocortin genes and co-operates with Pit-1 in activation of the growth hormone and prolactin genes. Pituitary Lim (P-Lim) protein also acts independently on the alpha-GSU gene, and acts in concert with Pit-1 to activate other genes. A fourth protein, termed the 'Prophet of Pit-1', or Prop-1, is the recently discovered cause of Ames dwarfism in mice. This paired-like protein is necessary for the subsequent expression of Pit-1 in somatotrophs, lactotrophs and thyrotrophs. Any or all of the newly discovered pituitary genes are candidates for mutations causing hypopituitarism in humans. As several are expressed transiently in tissues other than the pituitary during organogenesis, the phenotypes produced by mutations in these genes may prove to be complex.

Animals↗

PubMed1997

Effects of contraceptive estrogen and progestin on the atherogenic potential of plasma LDLs in cynomolgus monkeys.

This study was designed to determine the effect of oral contraceptive treatment (estrogen and progestin), alone or in combination, on LDL composition and atherogenic potential in cynomolgus monkeys fed an atherogenic diet. Groups (n = 8 each) of monkeys were untreated (control) or treated with ethinyl estradiol (EE), levonorgestrel (LNG); or triphasic oral contraceptive (EE+LNG) for 1.5 years before plasma LDLs were isolated for characterization. Total plasma cholesterol concentrations were unaffected by the treatments. LDL particle size (measured as LDL molecular weight, g/mumol) was significantly smaller, in the EE (4.61 +/- 0.09) and EE+LNG (4.43 +/- 0.09) treatment groups compared with the control (4.99 +/- 0.09) or LNG (5.29 +/- 0.17) groups and contained fewer molecules of free and esterified cholesterol. Both the EE and EE+LNG groups had significantly less cholesterol and apolipoprotein B distributed in the d = 1.015 to 1.025 g/mL subfraction and correspondingly more in the d = 1.025 to 1.035 g/mL subfraction of LDL compared with the control and LNG groups. The apolipoprotein E content (molecules/particle) of LDL was significantly less in the EE (0.35 +/- 0.1) and EE+LNG (0.28 +/- 0.1) groups compared with the control (0.86 +/- 0.2) and LNG (0.99 +/- 0.2) groups, and this trend was apparent in all three LDL subfractions. The atherogenic potential of LDL was tested using an in vitro binding assay to arterial proteoglycans. Twice as much LDL bound to arterial proteoglycans in the LNG group (11.3 +/- 1.8% of total LDL cholesterol in the incubation) compared with the control (6.4 +/- 1.9%), EE (5.5 +/- 1.5%), or EE+LNG (5.2 +/- 1.2%) groups. We conclude that EE and EE+LNG treatment alters the composition of LDL toward a less atherogenic particle that is smaller and more dense, contains less cholesterol and less apolipoprotein E, and is less reactive with arterial proteoglycans compared with LNG treatment. The inclusion of EE in the triphasic oral contraceptive treatment was sufficient to negate the potentially atherogenic effects of LNG on LDL composition.

Animals↗

PubMed1997

Pituitary adenomas: screening for G alpha q mutations.

Mutant, guanosine triphosphatase-deficient, alpha-subunits of the G protein, Gs, gsp ocogene have been discovered in 40% of GH-secreting pituitary adenomas. Therefore, we hypothesized that a novel G protein class, G alpha q, involved in pituitary signal transduction, might be involved in pituitary tumorigenesis. Recombinant mutations of G alpha q result in constitutive activation of phospholipase C and have transforming activity. Therefore, we screened tumor samples from 37 pituitary adenomas for the presence of activating mutations of the G alpha q gene. Importantly, our sample contains 8 FSH and LH adenomas. In the pituitary gland, FSH and LH are linked to the GnRH-G alpha q signaling cascade, making these tumors a logical choice for screening for G alpha q mutations. Complementary DNA (cDNA) was synthesized by RT-PCR with G alpha q specific primers to exclude pseudogene transcripts. Fragments of G alpha q cDNA-encompassing residues (Arg183, Gln209) were screened by single-strand conformation polymorphism and then sequenced in both directions. No mutations were detected. We conclude that mutations in these regions of the G alpha q cDNA occur infrequently, if at all, in human pituitary adenomas. Alternative mechanisms underlying pituitary tumorigenesis should be explored.

Adenoma↗

PubMed1997

Clinical, biochemical, and molecular investigations of a genetic isolate of growth hormone insensitivity (Laron's syndrome).

We have characterized the GH receptor mutation that is responsible for extreme short stature and GH insensitivity in a Bahamian genetic isolate. Heights of affected individuals ranged from -4.0 to -6.3 SD. Like others with Laron's syndrome, they had normal to high serum GH concentrations and low serum insulin-like growth factor I concentrations. Circulating levels of GH-binding protein activity were below limits of detection. Amplification of exons 2-7 and screening with single strand conformational polymorphism analysis located an abnormality in exon 7. Sequencing identified homozygosity for a C to T transition in the third position of codon 236. Reverse transcription and PCR amplification of complementary DNA from lymphocytes showed that this same sense mutation generated a new splice donor site 63 bp 5' to the normal exon 7 splice site. This novel site was used to the exclusion of the normal site in homozygotes. Both normal and variant messenger ribonucleic acid species were detected in heterozygotes. The predicted protein lacks 21 amino acids, including those defining the WS-like motif of the GH receptor extracellular domain. The high frequency of Laron's syndrome in this isolated island population probably reflects the introduction of the G236 splice mutation by a settler early in the 300-yr history of English settlement.

Adolescent↗

PubMed1997

Long-chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine decrease the stability of recombinant high density lipoprotein apolipoprotein A-I and the activation energy of the lecithin:cholesterol acyltransferase reaction.

The lecithin:cholesterol acyltransferase (LCAT) kinetics and activation energy and the stability of apolipoprotein A-I (apoA-I) were investigated using recombinant HDL (rHDL) containing phosphatidylcholine (PC), [3H]cholesterol, and apo A-I. The PC component of the rHDL contained sn-1 16:0 and sn-2 18:1 (POPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC) or 10% of the respective PC species and 90% sn-1 18:1, sn-2 16:0 PC ether (OPPC ether). The appVmax of the rHDL containing 100% PC varied 10-fold and was ordered POPC > PEPC > PAPC > PDPC, whereas the appKm values varied 19-43 microns PC. The ether-containing rHDL had appVmax values 17-40% of their respective 100% PC rHDL, but maintained the same rank order. The activation energy of LCAT was lower for rHDL containing long chain polyunsaturated fatty acids (PUFA) compared to rHDL containing 100% POPC or 10% PC/90% OPPC ether. The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.2-16 M) compared to those containing 100% POPC or 10% PC/90% OPPC (2.2-2.4 M) and there was a strong correlation (r = 0.71) between LCAT activation energy and the stability of apoA-I (i.e., D1/2). We conclude that long chain PUFA in the sn-2 position of PC decreases the catalytic efficiency of LCAT, the activation energy of the LCAT reaction and the stability of apoA-I on the rHDL particles. The strong association between rHDL apoA-I stability and LCAT activation energy suggests that the temperature-dependent step of the LCAT reaction may be sensitive to the strength of the interaction of apoA-I with rHDL PC.

Apolipoprotein A-I↗

PubMed1997

Influence of vesicle surface composition on the interfacial binding of lecithin:cholesterol acyltransferase and apolipoprotein A-I.

Interfacial binding affinities and capacities of lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein A-I (apoA-I) for surfaces of different phosphatidylcholine (PC) composition, cholesterol content, and apolipoprotein content were measured with a vesicle model system. Native polyacrylamide gel electrophoresis was used to separate free protein from vesicle-bound protein. ApoA-I was isolated from human plasma and radiolabeled with iodine, whereas radiolabeled LCAT was purified from the media of Chinese hamster ovary cells that were transfected with human LCAT cDNA and incubated in the presence of [35S] cysteine and methionine. Bound and free radiolabeled LCAT and apoA-I were quantified by phosphorimage analysis. ApoA-I binding was not influenced by cholesterol content (14 mole%) but was influenced by the PC fatty acyl composition of the vesicle. PC species containing long chain, polyunsaturated fatty acids (PUFA) in the sn-2 position resulted in increased binding affinity (Kd = 75-177 nM) but reduced capacity (0.1-0.3 apoA-I/ 1000 PC) in comparison to sn-1 palmitoyl, sn-2 oleoyl PC (POPC, 750 nM and 1.4 apoA-I/1000 PC). LCAT binding affinity to POPC (2190 nM) was stronger in the presence of cholesterol (530 nM), and LCAT binding capacity was reduced (2.63 and 0.6 molecules LCAT/1000 PC, respectively). In comparison to POPC, LCAT binding affinity to sn-1 palmitoyl, sn-2 arachidonyl PC was stronger (611 nM) and binding capacity was reduced (0.7 LCAT/1000 PC). LCAT binding affinity and capacity to sn-1 palmitoyl, sn-2 eicosapentaneoyl PC (2041 nM, and 2.5 LCAT/1000 PC) were similar to those observed for POPC. We conclude that vesicle surface PC fatty acyl composition and cholesterol content significantly influence LCAT and apoA-I interfacial binding and therefore may alter LCAT enzymatic activity.

Animals↗

PubMed1996

Effects of hormone replacement modalities on low density lipoprotein composition and distribution in ovariectomized cynomolgus monkeys.

This study was designed to determine the effect of several hormone replacement therapies on LDL size, density, heterogeneity, and composition in surgically postmenopausal cynomolgus monkeys fed an atherogenic diet. Groups (n = 5 each) of ovariectomized cynomolgus monkeys were untreated (control), or treated with conjugated equine estrogens, medroxyprogesterone acetate (progesterone), combined estrogen-progesterone, or tamoxifen for 9 weeks. There were no differences among treatment groups in total plasma, LDL, or HDL cholesterol or triglyceride concentrations. Plasma LDL were isolated by ultracentrifugation and size exclusion chromatography and subfractionated by density gradient centrifugation for subsequent chemical analysis. Estrogen treatment was associated with significantly smaller (measured as LDL molecular weight, 3.9 +/- 0.2 g/mu mol) and denser plasma LDL (1.034 g/ml peak density) compared with control (4.5 +/- 0.1 g/mu mol; 1.030 g/ml peak density) or progesterone-treated animals (4.6 +/- 0.2; 1.029 g/ml peak density). LDL from the estrogen group were relatively enriched in protein and triglyceride and poor in cholesteryl ester and apolipoprotein F (apoE) compared to the control group. Triglyceride enrichment with estrogen treatment occurred predominantly in the lighter, larger LDL subfractions (d = 1.015-1.025 g/ml), which were reduced in concentration (26 +/- 10 mg cholesterol/dl) compared to control (61 +/- 19 mg/dl) or progesterone treated animals (67 +/- 16 mg/dl). Combined estrogen-progesterone or tamoxifen treatment resulted in changes in LDL that followed the same trend as those observed with estrogen treatment. We conclude that short-term estrogen treatment of ovariectomized cynomolgus monkeys results in changes in plasma LDL size, density, and composition while having no apparent effect on overall plasma lipid concentrations.

Administration, Oral↗

PubMed1996

Optic nerve hypoplasia: absence of posterior pituitary bright signal on magnetic resonance imaging correlates with diabetes insipidus.

PURPOSE: Magnetic resonance imaging has been used to examine children with optic nerve hypoplasia for pituitary abnormalities who may be at risk for anterior pituitary hormonal deficiencies. We correlated the sellar and optic pathway anatomic findings on magnetic resonance imaging in children with optic nerve hypoplasia with findings from their endocrinologic and ophthalmologic examinations to determine whether magnetic resonance imaging findings predict anterior and posterior pituitary dysfunction. METHODS: A retrospective review identified five children with optic nerve hypoplasia and endocrinopathy who also underwent high resolution volumetric magnetic resonance imaging. RESULTS: All children had severe bilateral optic nerve hypoplasia and anterior pituitary hormone deficiencies. Three children had no recognizable intrasellar or ectopic posterior pituitary bright spot on magnetic resonance imaging; all had clinical evidence of diabetes insipidus. Two patients with a recognizable but ectopic posterior pituitary did not have diabetes insipidus. CONCLUSION: Children with optic nerve hypoplasia and no recognizable posterior lobe of the pituitary gland on magnetic resonance imaging are at risk for both anterior and posterior pituitary dysfunction, whereas those with a posterior lobe on magnetic resonance imaging appear to have intact posterior pituitary function.

Diabetes Insipidus↗

PubMed1996

Mechanisms responsible for dominant expression of human growth hormone gene mutations.

Point mutations of the donor splice site of intron 3 of the human GH-1 gene cause autosomal dominant inherited isolated growth hormone deficiency (IGHD II). The mechanism by which a defect in one GH-1 allele results in GH deficiency is obscure. Previously reported reverse transcription-nested PCR data suggested an overexpression of the mutant GH-1 allele. We employed alternative methods to determine the relative expression of mutant (C for G at +1 of intron 3) and normal GH-1 allele. The use of a second round PCR primer bridging exons 2 and 3 and specific for normal GH-1 messenger ribonucleic acid (mRNA) indicated equal quantities in mutant and control cells. Large scale messenger RNA extraction from Epstein-Barr virus-transformed lymphoblasts permitted assay by ribonuclease protection. In normal pituitary, there were three GH-1 mRNA species. The variant lacking exon 3 comprised 5% of the total GH-1 mRNA. The proband's lymphoblasts contained equal amounts of mRNA with and without exon 3. Only normal GH-1 mRNA was detected in controls. Secreted GH, measured by enzyme-linked immunosorbent assay was present in equal concentrations in media from normal and mutant cells. Thus, GH-1 mRNA lacking exon 3 was expressed in proportion to the dosage of the mutant gene, and dominant effects on GH secretion were not observed in lymphoblasts. These findings are compatible with a dominant negative mechanism involving interaction between normal and mutant proteins in secretory vesicles of somatotropes.

Adolescent↗

PubMed1996

Glycosylation structure and enzyme activity of lecithin:cholesterol acyltransferase from human plasma, HepG2 cells, and baculoviral and Chinese hamster ovary cell expression systems.

The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT), media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.

Amidohydrolases↗

PubMed1996

High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression.

To facilitate the investigation of apoA-I structure:function relationships as they relate to LCAT activation and lipid binding, we have developed an apoA-I baculoviral expression and purification system that yields milligram quantities of wild-type or mutant proapoA-I. Baculovirus-infected Sf-9 cells, grown in suspension, were found to secrete high levels of human wild-type (40-50 mg/l) or mutant apoA-I protein (1-38 mg/l), which was determined to be > 95% pure following a two-step purification procedure. In the case of wild-type apoA-I, ELISA showed that approximately 13-18% of the total protein secreted into the culture medium was apoA-I. To isolate pure protein from culture medium, 72 h post-infection medium was subjected to preparative reverse phase high performance liquid chromatography (HPLC), followed by DEAE ion-exchange chromatography. Purity and molecular size determination of wild-type proapoA-I protein was verified by SDS polyacrylamide gel electrophoresis, electrospray mass spectrometry, and N-terminal sequencing. In addition, recombinant discoidal apoA-I:phospholipid complexes prepared from wild-type or plasma apoA-I showed similar particle size and LCAT activation properties. To fully characterize the utility of this expression system, the expression levels of various mutant apoA-I proteins were compared to wild-type. Despite a lower production level seen with selected apoA-I mutants, milligram quantities of these purified mutant proteins were also obtained. In summary, we show that baculovirus-derived wild-type proapoA-I shows properties similar to plasma apoA-I relative to recombinant HDL formation, LCAT reactivity, and alpha-helical content. In addition, we show that a variety of mutant forms of human proapoA-I can be expressed and purified in abundant quantity from baculoviral-infected Sf-9 cells.

Animals↗

PubMed1996

Low density lipoprotein particle size and core cholesteryl ester physical state affect the proton NMR magnetic environment of fatty acid methylene and methyl nuclei.

Recent studies using proton NMR to quantify plasma lipoprotein concentrations have shown that lipoprotein particle size affects the chemical shift of methyl and methylene resonances. The purpose of this study was to investigate the interrelationship of low density lipoprotein (LDL) size and cholesteryl ester (CE) physical state on the chemical shift of methyl and methylene protons. LDL were isolated from the plasma of nonhuman primates fed diets containing lard or fish oil to result in a wide range of LDL particle sizes and CE core transition temperatures for NMR analysis. The proportion of nuclei in the fluid state in LDL at different temperatures, measured as proton NMR peak intensity, paralleled the melting profile of LDL CE determined by differential scanning calorimetry. At 37 degrees C the linewidths of several resolvable fatty acyl resonances, but not the choline methyl resonance, were significantly less in the fish oil LDL, indicating a less restrictive environment along the fatty acyl chain. The (CH2)n and t-CH3 resonances demonstrated an upfield shift (i.e., smaller chemical shift) with increasing temperature for both diet groups, but the peaks for fish oil LDL were always upfield from those of the lard LDL regardless of temperature. No change in the chemical shift of the choline CH3 resonance was observed as a function of dietary fat or temperature. Above the transition temperature for LDL CE there was a significant positive correlation between LDL size and the chemical shift of the (CH2)n and t-CH3 resonances. This relationship was not statistically significant for the same LDL below the Ce transition temperature. We conclude that LDL CE must be fully melted to obtain accurate results for LDL particle size distribution as well as mass quantification using portion NMR spectroscopy.

Animals↗

PubMed1996

Plasma cholesteryl ester transfer activity is modulated by the phase transition of the lipoprotein core.

Previous studies have shown that lipid transfer protein (LTP) activity is strongly temperature dependent, demonstrating a dramatic rise in activity near 37 degrees C. We have investigated the origin of this rapid rise in LTP activity. LTP-mediated transfers of radiolabeled cholesteryl ester (CE) from LDL to HDL, HDL to LDL, LDL to biotin-LDL, HDL to biotin-HDL, and between liposomes were determined as a function of assay temperature. Only assays containing LDL demonstrated this rapid rise in CE transfer activity. In contrast, TG transfer was almost linear with assay temperature. As human LDL CE undergoes a thermal phase transition near 37 degrees C, we investigated whether the rapid rise in CE transfer was dependent on this transition. Monkey LDL were isolated from animals consuming diets containing cholesterol and enriched in saturated, monounsaturated, or polyunsaturated fatty acids. With these LDL as substrate, the CE transfer between 21 degrees and 49 degrees C could be described by two straight lines, the intersection of which defined the inflection temperature. Among eight LDL samples, the inflection temperature was highly correlated with the CE phase transition determined by differential scanning calorimetry (r2 = 0.86). Both calorimetry and CE transfer activity inflection values were correlated with the saturated + monoene/polyene ratio of the LDL cholesteryl esters (r2 = 0.733 and 0.612, respectively). For LDL with inflection temperatures below 37 degrees C, CE transfer activity at 37 degrees C increased 10-14% for each 1 degree C decrease in the inflection temperature. We conclude that LTP activity is markedly affected by the physical state of the core CE. Diets rich in saturated fatty acids may result in LDL that are poor LTP substrates, which may hinder LTP's ability to promote normal lipoprotein remodeling.

Animals↗

PubMed1995

Dietary polyunsaturated fat modifies low-density lipoproteins and reduces atherosclerosis of nonhuman primates with high and low diet responsiveness.

We tested the hypothesis that an increased content of n-6 polyunsaturated fatty acids (principally linoleic acid) in an atherogenic diet of nonhuman primates would decrease atherosclerosis by modifying the composition and decreasing the concentration of plasma low-density lipoprotein (LDL). A species readily susceptible to diet-induced atherosclerosis (cynomolgus monkey) was compared with a less-susceptible species (African green monkey) with dietary cholesterol concentration and saturated or polyunsaturated fat (40% of energy) as variables. In both species, cholesterol concentrations in whole plasma, LDL, and high-density lipoprotein (HDL) were 20-30% lower when polyunsaturated fat was fed, whereas dietary cholesterol increased LDL cholesterol three- to fourfold. LDL was enriched in cholesteryl oleate when saturated fat and cholesterol were fed. Dietary linoleic acid prevented cholesteryl oleate enrichment and promoted cholesteryl linoleate accumulation in LDL. At the same plasma cholesterol concentration, cynomolgus monkeys had higher LDL cholesterol and lower HDL-cholesterol concentrations than did African green monkeys. LDL particle size was significantly (P < 0.001) larger in the group of cynomolgus monkeys fed polyunsaturated fat but tended to be smaller in African green monkeys fed polyunsaturated fat. Dietary polyunsaturated fat protected against coronary artery atherosclerosis in both species. Thus, LDL particle size, per se, was not atherogenic; instead, coronary artery atherosclerosis and cholesteryl oleate enrichment of LDL were more highly correlated. This outcome suggests that information about LDL composition may be more important for understanding the pathogenesis of atherosclerosis than previously suspected.

Animals↗