Ligand-binding properties of albumin Parklands: Asp365----His.

An albumin variant, isolated from the plasma of a patient with bisalbuminemia, was compared with albumin A from the same patient for binding of long-chain fatty acids and bilirubin. No differences in binding of [14C]palmitate, cis-parinaric acid or bilirubin could be detected for the variant form. These results suggest that the region adjacent to residue 365 is unlikely to be part of a major binding site for any of these ligands.

Plasma sphingomyelin level as a risk factor for coronary artery disease.

Only a fraction of the clinical complications of atherosclerosis are explained by known risk factors. Animal studies have shown that plasma sphingomyelin (SM) levels are closely related to the development of atherosclerosis. SM carried into the arterial wall on atherogenic lipoproteins may be locally hydrolyzed by sphingomyelinase, promoting lipoprotein aggregation and macrophage foam cell formation. A novel, high-throughput, enzymatic method to measure plasma SM levels has been developed. Plasma SM levels were related to the presence of coronary artery disease (CAD) in a biethnic angiographic case-control study (279 cases and 277 controls). Plasma SM levels were higher in CAD patients than in control subjects (60+/-29 versus 49+/-21 mg/dL, respectively; P:<0. 0001). Moreover, the ratio of SM to SM+phosphatidylcholine (PC) was also significantly higher in cases than in controls (0.33+/-0.13 versus 0.29+/-0.10, respectively; P:<0.0001). Similar relationships were observed in African Americans and whites. Plasma SM levels showed a significant correlation with remnant cholesterol levels (r=0.51, P:<0.0001). By use of multivariate logistic regression analysis, plasma SM levels and the SM/(SM+PC) ratio were found to have independent predictive value for CAD after adjusting for other risk factors, including remnants. The odds ratio (OR) for CAD was significantly higher for the third and fourth quartiles of plasma SM levels (OR 2.81 [95% CI 1.66 to 4.80] and OR 2.33 [95% CI 1.38 to 3. 92], respectively) as well as the SM/(SM+PC) ratio (OR 1.95 [95% CI 1.10 to 3.45] and OR 2.33 [95% CI 1.34 to 4.05], respectively). The findings indicate that human plasma SM levels are positively and independently related to CAD. Plasma SM levels could be a marker for atherogenic remnant lipoprotein accumulation and may predict lipoprotein susceptibility to arterial wall sphingomyelinase.

High levels of Lp(a) with a small apo(a) isoform are associated with coronary artery disease in African American and white men.

Elevated levels of lipoprotein(a) [Lp(a)] and the presence of small isoforms of apolipoprotein(a) [apo(a)] have been associated with coronary artery disease (CAD) in whites but not in African Americans. Because of marked race/ethnicity differences in the distribution of Lp(a) levels across apo(a) sizes, we tested the hypothesis that apo(a) isoform size determines the association between Lp(a) and CAD. We related Lp(a) levels, apo(a) isoforms, and the levels of Lp(a) associated with different apo(a) isoforms to the presence of CAD (>/=50% stenosis) in 576 white and African American men and women. Only in white men were Lp(a) levels significantly higher among patients with CAD than in those without CAD (28.4 versus 16.5 mg/dL, respectively; P:=0.004), and only in this group was the presence of small apo(a) isoforms (<22 kringle 4 repeats) associated with CAD (P:=0.043). Elevated Lp(a) levels (>/=30 mg/dL) were found in 26% of whites and 68% of African Americans, and of those, 80% of whites but only 26% of African Americans had a small apo(a) isoform. Elevated Lp(a) levels with small apo(a) isoforms were significantly associated with CAD (P:<0.01) in African American and white men but not in women. This association remained significant after adjusting for age, diabetes mellitus, smoking, hypertension, HDL cholesterol, LDL cholesterol, and triglycerides. We conclude that elevated levels of Lp(a) with small apo(a) isoforms independently predict risk for CAD in African American and white men. Our study, by determining the predictive power of Lp(a) levels combined with apo(a) isoform size, provides an explanation for the apparent lack of association of either measure alone with CAD in African Americans. Furthermore, our results suggest that small apo(a) size confers atherogenicity to Lp(a).

Cholesterol lowering with pravastatin improves resistance artery endothelial function: report of six subjects with normal coronary arteriograms.

STUDY OBJECTIVES: Improvement in coronary artery endothelial function has been demonstrated after cholesterol lowering in hypercholesterolemic patients with significant atherosclerosis. However, to our knowledge, no previous study has shown improvement in resistance artery function in subjects with normal coronary arteries after cholesterol lowering. The purpose of our study was to investigate the effect of cholesterol lowering with pravastatin on coronary resistance artery endothelial function in the setting of angiographically normal coronary arteries. METHODS: Invasive testing of coronary endothelial and vasomotor function was performed at baseline and after 6 months of pravastatin treatment in six patients with normal coronary arteriograms. RESULTS: After 6 months of pravastatin treatment, low-density lipoprotein cholesterol level dropped from 157+/-11 to 117+/-8 mg/dL (p = 0.02) and percent increase in coronary blood flow after acetylcholine improved from 97+/-13% to 160+/-16% (p = 0.01). There was a trend (p = 0.17) toward enhanced epicardial dilation in response to acetylcholine after pravastatin treatment when compared with the baseline study. CONCLUSIONS: Our study demonstrates significant improvement in coronary resistance artery endothelial function after 6 months of cholesterol lowering with pravastatin in six subjects presenting with chest pain who were found to have normal coronary arteriograms. A trend toward improved epicardial vasomotion was also observed.

Variation of lipids and lipoproteins in premenopausal women compared with men and postmenopausal women. DELTA (Dietary Effects on Lipoproteins and Thrombogenic Activity) Investigators.

Numerous studies have reported cyclic fluctuations in lipids and lipoproteins as a function of the phase of the menstrual cycle. However, the reported patterns are quite variable and have led to an unclear picture of the influence of the menstrual cycle on the variability of lipids, and hence of the role of the menstrual cycle phase in the interpretation of serum lipids for premenopausal women. As part of the DELTA Study (Dietary Effects on Lipoproteins and Thrombogenic Activity), we evaluated the cyclic variation of circulating lipids and lipoproteins in 39 premenopausal women and compared intraindividual variances in these women, 18 postmenopausal women, and 46 men under conditions of tight dietary control. Cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, triglyceride, apolipoproteins A-1 (apo A-1) and B-100 (apo B-100), and lipoprotein (a) [Lp(a)] all demonstrated cycling in the premenopausal women. However, the observed cycling accounts for only a small fraction of the total biologic variability of lipids in premenopausal women. The magnitude of total intraindividual variability based on coefficient of variation (CV) for these lipids in premenopausal women (CV, 4% to 8.1%) was similar to that found for men (CV, 4.3% to 9.1%) and for postmenopausal women (CV, 3.7% to 6.7%). These results suggest that protocols for screening and monitoring of serum lipids in premenopausal women need not differ from those used for men or postmenopausal women.

Fatty acid carbon isotope ratios in humans on controlled diets.

Carbon stable isotope ratios for six serum fatty acids (FA) are reported for human subjects on controlled fat diets to determine the range of natural isotope abundance and to demonstrate the leveling effect of a well-controlled diet. Twenty-nine subjects were randomly assigned to one of three controlled diets containing high, medium, or low fat. Diets were consumed for 8 wk. Serum samples were collected at baseline (0), 5, 6, 7, and 8 wk. FA were extracted and methylated. Isotope ratios were analyzed by high-precision gas chromatography combustion-isotope ratio mass spectrometry. At baseline, mean delta 13C for 16:0b, 16:1a, 18:0a, 18:1c,18:2n-6d and 20:4n-6bc were -24.1, -21.7, -21.6, -25.6, -29.6, and -25.0/1000, respectively, with an average standard deviation of 1.9/1000. Most delta 13C decreased during the diet period and appeared to have stabilized by week 5 at -25.3, -21.9, -22.3, -26.5, -30.1, and -24.5/1000, respectively. Between-subjected variability decreased from 1.74 to 1.20/1000 on the controlled diets. Measurement variability was 0.53/1000. The within-subject variability during weeks 5-8 was 0.57/1000 (range of 0.32-0.84/1000), showing a minimum biological fluctuation on controlled diets. There was no diet group effect on delta 13C of serum FA. Except for 18:2, the delta 13C of experimental diets was lower than that of serum FA, consistent with observations in animals. These data show that carbon isotope ratios stabilize in response to controlled diets within 5 wk, reflecting the isotope ratio of their dietary source, and establish isotope ratio fluctuations for endogenous compounds for future studies.

Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling.

Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.

Spurious cell surface receptors: inadequate correction for saturable, nonspecific binding mimics receptor binding.

Methods commonly used for the characterization of receptors on cell surfaces may be subject to adsorption artifacts that mimic the action of receptors even when no receptors are present. Traditional techniques such as incubation in the presence of an unrelated protein to minimize adsorption or extensive washing to eliminate nonspecific binding may be inadequate to ensure that the observed binding is due to a cell membrane--ligand interaction. In this paper three serum proteins, albumin, transferrin, and immunoglobulin G, are shown to exhibit behavior suggestive of receptor-mediated binding even in the absence of cells. Two types of control studies are suggested to establish that observed binding is attributable to interaction with cells.

Kinetics of bilirubin binding to bovine serum albumin and the effects of palmitate.

The binding of bilirubin to bovine serum albumin has been determined to be a second order process, first order in each reactant. Formation of the bilirubin-albumin complex was observed as an increase in absorbance at 480 nm by stopped flow spectrophotometry. The effect of long chain fatty acid on the binding of bilirubin was investigated at palmitic acid levels of 0 to 7 mol/mol of albumin at 37 degrees and pH 7.4. The second order rate constant is 1.5 X 10(6) M-1 S-1 when the palmitate/albumin ratio is less than 5 and drops to 0.5 X 10(6) M-1 S-1 when the palmitate/albumin ratio reaches 6. The equilibrium association constant for the bilirubin-albumin complex is 5.5 X 10(6) M-1 when the palmitate/albumin ratio is less than 1, 18 X 10(6) M-1 when the palmitate/albumin ratio is between 1 and 5, and 3 X 10(6) M-1 when the palmitate/albumin ratio is greater than 5. The mechanism of the effect of palmitate varies with the total palmitate level. At low molar ratios, addition of palmitate has no effect on the association rate but decreases the dissociation rate. At high molar ratios, addition of palmitate decreases the association rate and increases the dissociation rate. It is suggested that palmitate affects the ability of the protein to undergo the conformational changes needed to accommodate bilirubin.

Bromcresol purple dye-binding methods underestimate albumin that is carrying covalently bound bilirubin.

We used automated and manual versions of two commonly used dye-binding methods for albumin to quantify albumin that had been modified by covalent addition of bilirubin. Dye-binding methods based on reaction with bromcresol purple underestimated the modified albumin by as much as 29%. Dye-binding methods based on reaction with bromcresol green were unaffected by the presence of covalently attached bilirubin. Neither method was affected by the presence of noncovalently bound bilirubin.

Oxalate removal by hemodialysis in end-stage renal disease.

Because of mounting evidence of precipitation of calcium oxalate in the soft tissues of patients with end-stage renal disease (ESRD) on maintenance hemodialysis, the plasma oxalate concentrations and calculated dialysis removal of oxalate were studied in seven patients without evidence of either primary or absorption hyperoxaluria prior to ESRD. A reversed-phase high-pressure liquid chromatographic method was developed to quantitate serum oxalate. Mean value +/- SE in four healthy controls was 28 +/- 5 mumol/L, and in the seven patients it was 187 +/- 15 mumol/L predialysis and 89 +/- 11 mumol/L postdialysis. Oxalate deposition in the soft tissues of ESRD patients is the consequence of sustained hyperoxalemia. Oxalate removal by dialysis was calculated from the four-hour oxalate clearance. Since the ionic radii of phosphate and oxalate are similar, total oxalate clearance was calculated midpoint of dialysis. Mean oxalate removal/dialysis was 3.01 +/- 0.283 mmol. On a daily basis this value was 1.645 +/- 0.155 mmol, which is about threefold the normal oxalate excretion rate. It is not significantly different from the excretion rate in absorption oxalurias but is less than that in primary hyperoxaluria. Therefore, it is concluded that hyperoxalemia in ESRD results from loss of renal excretion, failure of hemodialysis to remove enough oxalate to maintain a normal serum concentration, and increased intestinal absorption of oxalate and/or increased endogenous production.

The albumin receptor effect may be due to a surface-induced conformational change in albumin.

To determine whether equilibrium binding between albumin and hepatocytes involves a cell surface receptor for albumin, we incubated freshly isolated rat hepatocytes with 125I-albumin and determined the amount of albumin associated with the cells as a function of the total albumin concentration. The resulting two-phase binding curve showed the rat albumin-hepatocyte interaction to consist of a saturable binding interaction with a dissociation constant of 1.1 microM and 2 X 10(6) sites/cell in addition to a weak, nonsaturable binding interaction. However, the saturable binding of albumin to hepatocytes did not appear to result from the presence of an albumin receptor on the cell surface; the interaction was the same for different species of albumin, for chemically modified albumins, and for fragments of albumin representing mutually exclusive domains of the molecule. The saturable binding was, instead, found to involve a subpopulation of albumin with an enhanced affinity for the cell surface. We show that this subpopulation of albumin is generated upon contact with either solid surfaces or cell surfaces and can be transferred from one surface to another. We propose that the two-phase Scatchard binding curve and the "albumin receptor effect" reflect two populations of albumin that bind to the cell surface with different affinities rather than one population of albumin that binds to two classes of binding sites.

Preparation of fragments 505-582 and 505-573 of bovine serum albumin.

Peptic fragment 505-582 of bovine serum albumin, the "Phe" fragment, has been found useful in several laboratories for studies of antigenic sites, ligand binding and metabolism. It contains the entire carboxyterminal loop, Loop 9, of the albumin molecule. We present an improved preparation of this peptide. Peptide 505-582 is obtained in about 9% yield, along with a similar yield of the closely associated peptide 505-573 arising from a second peptic cleavage. The unusual ultraviolet absorption spectrum of these peptides shows triple maxima near 259 nm reflective of the high phenylalanine content and the total absence of tyrosine and tryptophan.

Role of a central laboratory in implementing national cholesterol education panel guidelines in rural practices: model system for managed care.

We describe the use of a central laboratory to identify patients who may be candidates for a hypercholesterolemia treatment program and to direct their referral into this program. The laboratory, providing service for 16 medical practices in a rural area of upstate New York, served as the entry point to the treatment program for those patients with serum cholesterol > or = 5.18 mmol/L. This treatment program, designed to follow the National Cholesterol Education Program Adult Treatment Panel Guidelines, was provided by the lipid referral center staff, including a registered dietitian and a lipid specialist. After introduction of this program, 52% of eligible patients received nutritional counseling for hypercholesterolemia, compared with only 29% in usual care settings. This program represents an enhanced role for laboratories in the implementation of treatment protocols typical of those adopted by managed care networks.

Immunochemical studies of fragments of bovine serum albumin.

Antigenic properties of 14 fragments of bovine albumin were measured using antisera to albumin and to two of its fragments. All seven fragments larger than 21,000 daltons formed immune precipitates. Although immune precipitates were not formed with smaller fragments, inhibition tests indicated the presence of antigenic sites on several of these fragments as well. The results predict the occurrence of six or more antigenic determinants and allow assignment of their positions in the parent molecule. These sites are distributed along the entire protein chain, with the sites of greatest antibody affinity situated in the COOH-terminal region. Evidence is presented that some sites are homologous, reacting with the same populations of antibodies, and that other sites are unique, binding to an exclusive population of antibodies.

Sequence of residues 400--403 of bovine serum albumin.

A large tryptic peptide of bovine serum albumin (residues 377--582) was subjected to 32 cycles of Edman degradation to determine the sequence of the last remaining unknown segment of this protein. Residues 400--403 were identified as gly-Phe-Gln-Asn. Amide assignments were also made at positions 388 (glutamine), 389 (asparagine), 391 (aspartic acid) and 392 (glutamine).

Fragments of bovine serum albumin produced by limited proteolysis. Conformation and ligand binding.

Twelve fragments of bovine serum albumin, isolated following limited tryptic or peptic hydrolysis, have been studied to define secondary structure and locate ligand-binding sites. Based on circular dichroism, the conformational pattern of albumin (68% alpha helix and 18% beta structure) is substantially retained by individual fragments, indicating that secondary configuration is locally determined and is not destroyed during the cleavage process nor during fragment purification. The strong bilirubin-binding site of bovine serum albumin is present in 3 of the 12 fragments. Residues 186-238 are common to the three fragments and absent from those fragments which do not bind bilirubin; consequently the strong bilirubin-binding site is suggested to involve this region. By similar reasoning, the presence of palmitate-binding sites in some fragments and not in others indicates that the three strongest sites for the binding of palmitate are located in the carboxyl-terminal two-thirds of the molecule. The first site (KA approximately 2 X 10(7) M-1) is suggested as residues 377-503; the second site (KA approximately 8 X 10(6) M-1), residues 239-306; the third site (KA approximately 2 X 10(6) M-1), residues 307-377. Bromocresol Green, a reagent used in the assay of ablumin, was bound by fragments rougly in proportion to their size but showed particular affinity for the region of the strong bilirubin-binding site. The fluorescent probe, 8-anilino-1-naphthalensulfonate, was in general bound by large fragments, supporting the concept that this ligand is held principally in clefts between domains of the macromolecule.

Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin.

Binding of 13C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin--two complementary fragments corresponding to the two halves of albumin and one fragment corresponding to the carboxyl-terminal domain--yielded unique patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding of the first 5 mol of oleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). Two of these resonances were seen upon addition of 1 or 2 mol of oleic acid to fragments representing either the carboxyl-terminal half (residues 307-582) or the carboxyl-terminal domain (residues 377-582); the third resonance was seen upon addition of 1 mol of oleic acid to the fragment representing the amino-terminal half (residues 1-306). The resonance patterns for the fourth and fifth moles of oleic acid added to albumin (secondary sites) could not be duplicated by addition of more oleic acid to individual fragments. These resonance patterns were generated, however, when the two complementary fragments were mixed in equimolar proportions to form an albumin-like complex with a reconstituted middle domain. Thus, two primary fatty acid binding sites are assigned to the carboxyl-terminal domain, one primary site is assigned to the amino-terminal half, and the secondary sites are assigned to the middle domain. This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domains.