Roberta G. Reed

Effects of Reducing Dietary Saturated Fatty Acids on Plasma Lipids and Lipoproteins in Healthy Subjects: The Delta Study, Protocol 1

Few well-controlled diet studies have investigated the effects of reducing dietary saturated fatty acid (SFA) intake in premenopausal and postmenopausal women or in blacks. We conducted a multicenter, randomized, crossover-design trial of the effects of reducing dietary SFA on plasma lipids and lipoproteins in 103 healthy adults 22 to 67 years old. There were 46 men and 57 women, of whom 26 were black, 18 were postmenopausal women, and 16 were men > or =40 years old. All meals and snacks, except Saturday dinner, were prepared and served by the research centers. The study was designed to compare three diets: an average American diet (AAD), a Step 1 diet, and a low-SFA (Low-Sat) diet. Dietary cholesterol was constant. Diet composition was validated and monitored by a central laboratory. Each diet was consumed for 8 weeks, and blood samples were obtained during weeks 5 through 8. The compositions of the three diets were as follows: AAD, 34.3% kcal fat and 15.0% kcal SFA; Step 1, 28.6% kcal fat and 9.0% kcal SFA; and Low-Sat, 25.3% kcal fat and 6.1% kcal SFA. Each diet provided approximately 275 mg cholesterol/d. Compared with AAD, plasma total cholesterol in the whole group fell 5% on Step 1 and 9% on Low-Sat. LDL cholesterol was 7% lower on Step 1 and 11% lower on Low-Sat than on the AAD (both P<.01). Similar responses were seen in each subgroup. HDL cholesterol fell 7% on Step 1 and 11% on Low-Sat (both P<.01). Reductions in HDL cholesterol were seen in all subgroups except blacks and older men. Plasma triglyceride levels increased approximately 9% between AAD and Step 1 but did not increase further from Step 1 to Low-Sat. Changes in triglyceride levels were not significant in most subgroups. Surprisingly, plasma Lp(a) concentrations increased in a stepwise fashion as SFA was reduced. In a well-controlled feeding study, stepwise reductions in SFA resulted in parallel reductions in plasma total and LDL cholesterol levels. Diet effects were remarkably similar in several subgroups of men and women and in blacks. The reductions in total and LDL cholesterol achieved in these different subgroups indicate that diet can have a significant impact on risk for atherosclerotic cardiovascular disease in the total population.

Plasma Sphingomyelin Level as a Risk Factor for Coronary Artery Disease

Abstract—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

Abstract—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).

Calcium Oxalate Microcrystalline-Associated Arthritis in End-Stage Renal Disease

Chronic renal failure is known to be associated with secondary hyperoxalemia and the deposition of calcium oxalate in visceral organs, bones, and cartilage. We report the identification of calcium oxalate crystals in the synovial fluid of three patients with chronic renal failure. In one patient, calcium oxalate crystals were also identified within synovium and cartilage. Crystals were pleomorphic and bipyramidal. Some crystals were rod-like and had positive birefringence, thus tending to be confused with calcium pyrophosphate dihydrate when observed with only compensated polarized light microscopy. In one patients asymptomatic effusions were associated with joint capsule calcification, but otherwise normal knee radiographs. The other two patients had bilateral knee pain, one having coexistent features of osteoarthritis and the other chondrocalcinosis. Samples of proliferative synovium, joint capsule, and cartilage from the patient with chondrocalcinosis showed abundant calcium oxalate crystals, and not calcium pyrophosphate dihydrate or calcium hydroxyapatite. Thus, radiographically typical chondrocalcinosis may be due to calcium oxalate. Joint disease in chronic renal failure may be associated with calcium oxalate as well as the previously recognized apatite deposition.

ApoE Genotype Does Not Predict Lipid Response to Changes in Dietary Saturated Fatty Acids in a Heterogeneous Normolipidemic Population

Recent studies have suggested that variations in apoE genotypes may influence the magnitude of plasma lipid changes in response to dietary interventions. We examined the ability of apoE genotype to predict plasma lipid response to reductions in percent of calories from total fat (TF) and saturated fat (SF) in a normolipidemic study population (n = 103) heterogeneous with respect to age, gender, race, and menopausal status. Three diets, an average American diet (34.3% TF, 15.0% SF), an AHA Step 1 diet (28.6% TF, 9.0% SF), and a low saturated fat (Low-Sat) diet (25.3% TF, 6.1% SF) were each fed for a period of 8 weeks in a three-way crossover design. Cholesterol was kept constant at 275 mg/d; monounsaturated and polyunsaturated fat were kept constant at approximately 13% and 6.5% of calories, respectively. Fasting lipid levels were measured during each of the final 4 weeks of each diet period. Participants were grouped by apoE genotype: E2 (E2/2, E2/3, E2/4); E3 (E3/3); E4 (E3/4, E4/4). Relative to the average American diet, both the Step 1 and Low-Sat diets significantly reduced total cholesterol, LDL cholesterol, and HDL cholesterol in all three apoE genotype groups. No evidence of a significant diet by genotype interaction, however, could be identified for any of the measured lipid and lipoprotein end points. Additional analysis of the data within individual population subgroup (men and women, blacks and whites) likewise provided no evidence of a significant diet by genotype interaction. Thus, in a heterogeneous, normolipidemic study population, apoE genotype does not predict the magnitude of lipid response to reductions in dietary saturated fat.

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.

Serum albumin stimulation of synaptosomal proline uptake partial identification of the active site

Abstract Synaptosomal uptake of proline is stimulated by serum albumin. Evidence is presented which indicates that peptide fragments isolated from the N-terminal region of serum albumin do not stimulate synaptosomal uptake of proline, while fragments derived from the C-terminal region have stimulatory capacities comparable to that of the parent albumin. The site responsible for the stimulatory effect has been tentatively located in the sequence region 377–504 of the albumin molecule.

Albumin immobilized on agarose as a tool for measuring ligand binding by proteins or peptides

Abstract Bovine serum albumin immobilized on agarose has been tested in competitive binding studies as a means of measuring the binding of cortisol, tryptophan, fatty acids, and bilirubin to a number of albumins and albumin fragments. Chemical coupling of albumin to agarose does not appear to alter the primary binding sites for most ligands and the degree of ligand binding by immobilized albumin is comparable to that by soluble albumin. Evaluation of ligand binding by a protein based on its competition with immobilized protein is suggested as a convenient procedure particularly well suited to proteins and ligands whose size precludes investigation by dialysis or whose instability demands a rapid procedure.

Immune recognition of serum albumin—XII. Evidence for time-dependent immunochemical cross-reactivity of subdomains 3, 6 and 9 of bovine serum albumin by quantitative immunoadsorbent titration studies

Abstract Antisera were raised in rabbits against bovine serum albumin (BSA) or against fragment 377–571 (domain 3) of BSA. Serial bleedings were obtained at different times after immunization. Fragments 115–184, 307–385 and 504–581 were isolated in pure form from BSA. These corresponded essentially to subdomains 3, 6 and 9 respectively of BSA. The capacity of each fragment to bind 125 I-labelled antibodies of rabbit anti-BSA or anti-domain 3 antisera was determined by a quantitative immunoadsorbent titration technique. With anti-BSA antisera from each rabbit, the maximum (plateau) amount of antibodies bound by each fragment was found to increase with time antisera are obtained after the first immunization. With antisera against domain 3, considerable amounts of antibodies were bound by subdomains 3, 6 and 9 and the fraction of cross-reacting antibodies also increased with time after the initial immunization. Antibodies against each subdomain fragment were isolated on fragment adsorbents and were investigated for their cross-reaction with the other two fragments. Specific antibodies against each subdomain were found to cross-react with the other two subdomains. The degree of cross-reaction increased remarkably with the duration of immunization and reached as high as 100% for the cross-reaction between subdomains 3 and 6. The results are consistent with an unusual functional equivalence of antigenic sites within the same protein molecule. This functional equivalence of the antigenic sites of albumin, which we had previously proposed, improves with time after immunization.

Immunochemistry of serum albumin—VIII: The antigenic reactivity of the third domain of bovine serum albumin resides in the last sub-domain. a dynamic examination of the change of antibody affinity and specificity☆

Abstract In a preceding paper, immunochemical and conformational studies on chemical derivatives of fragment 377–571 of bovine serum albumin (BSA) suggested that the immunochemical reactivity of this fragment (the third domain of the albumin molecule) resided entirely in the last sub-domain. In the present work, the immunochemical reactivities of fragments 377–571 and 504–581 with serial antisera to BSA were studied in detail. Antisera against BSA were raised in two rabbits and serial bleedings were obtained at different periods after the first immunization. The serial antisera were not mixed but were kept and studied separately. Immunoadsorbent titration studies showed that the plateau values of 125 I-antibody binding by the immunoadsorbents of the two fragments changed with time after the initial immunization but were, for a given antiserum, identical in all serial antisera from 15 days up to 398 days. However, in very early antisera (7 days) the larger fragment 377–571 possessed a higher immunochemical reactivity than the small fragment 504–581. The inhibitory activities of the two fragments towards the binding of 125 -albumin and 125 I-fragment 377–571 with serial antisera to BSA in a Farr assay were compared. The two fragments exhibited comparable inhibitory activities and, in addition, fragment 504–581 was able to inhibit completely the binding of 125 I-fragment 377–571. Acid dissociation studies of complexes of serial 125 I-antibodies on immunoadsorbents revealed that the affinity of the antibodies increased while their heterogeneity decreased with time after immunization. The results with serial antisera from each of the two rabbits show that although in very early (7 days) antisera, the larger fragment appears to carry some additional antigenic sites, the shared antigenic sites become completely immunodominant relatively early (15 days) after the first immunization.