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Purpose: To evaluate cardiovascular risk factors in Caucasian and African American HIV+ women undergoing treatment with HAART including a protease inhibitor (PI)., Method: Anthropometric measures and fasting blood samples were obtained from 32 Caucasian and 10 African American women. Serum was analyzed for glucose, insulin, and lipid levels., Results: The African American women were significantly older than the Caucasian women. Body mass index (BMI) was higher in the African American women, and 80% of the African American women and 47% of the Caucasian women were overweight. There were no significant differences in fasting insulin, fasting glucose, or HOMA-IR. However, African American women had significantly higher HDL levels, whereas Caucasian women had higher triglycerides and LDL. When age-matched women were compared, total as well as LDL cholesterol was significantly higher in the Caucasian women. The ratio of total cholesterol/HDL cholesterol was 3.4 +/- 1.1 in the African American women and 5.5 +/- 1.6 (p=.021) in the age-matched Caucasian women., Conclusion: The differences in lipid levels in HIV+ African American and Caucasian women were greater than those reported in the literature for normal women. Although the sample size is small, the data suggest that the effect of HIV infection and/or HAART on lipid levels may be different in Caucasian and African American women. Large-scale studies will be necessary to confirm these results and clarify the mechanisms involved.

Background: In a previously reported study, 21 women (propositi) who reported changes in body habitus during highly active antiretroviral therapy (HAART) were evaluated and compared with 21 women (comparison group) on HAART who did not report body habitus changes. Mean durations of HAART at baseline evaluation were 12.5 and 15.2 months for the propositi and comparison group, respectively., Objective: Follow-up of the propositi and comparison group was conducted to determine whether body habitus changes and lipid abnormalities are progressive, stable, or improved with time and alteration of the HAART regimen., Methods: Patients were evaluated by standardized interview, physical examination, body weight, body mass index, CD4 cell count, plasma HIV RNA levels, and lipid profiles., Results: Fourteen of 21 propositi were available for follow-up. The mean duration of HAART was 42.7 months; body habitus changes were stable in 10 of the 14 women. Thirteen of 21 women in the comparison group were available for follow-up after a mean duration of HAART of 38.5 months; 2 of the 13 women had developed body habitus changes at follow-up. In both groups, mean serum lipid values at follow-up remained elevated to levels associated with increased cardiovascular risk., Conclusions: Body habitus changes in women most often developed within 1 year of initiation of HAART. Changes were largely stable after 2.5 additional years of HAART. Only modest and inconsistent improvement was achieved with alteration in the HAART regimen. Serum lipid abnormalities evident within the first year of HAART were also stable with 2.5 additional years of therapy.

Twenty-one women (propositi) who expressed serious concerns about changes in body habitus during highly active antiretroviral therapy (HAART) were evaluated by thorough physical examination, anthropometric measurements, and serum lipid and endocrine assays. The same evaluations were carried out in a comparison group of 21 women who received HAART but did not complain of changes in habitus. No significant demographic differences were found between the propositi and the comparison group, nor were there significant differences in CD4 count or plasma viral load (PVL) between the two groups. Lipid analyses were also performed on plasma obtained prior to HAART from 12 of the women. The frequency of changes reported by the 21 propositi were increase in abdominal size (90%), increase in breast size (71%), weight gain of >5 kg (43%), peripheral fat wasting (43%), buttock fat wasting (38%) and development of cervicodorsal fat pad (19%). A subset of patients in the comparison group experienced increase in abdominal size (29%) and weight gain >5 kg (19%), but none experienced clinically detectable peripheral or buttock fat wasting, increased breast size, or development of cervicodorsal fat pads. Mean waist circumference, waist-to-hip ratios (WHR), body fat, and body mass index (BMI) were above the desirable range for women in both propositi and the comparison group. Levels of total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol associated with increased cardiovascular risk were found in 48%, 62%, 45%, and 33%. respectively, of the propositi, with similar findings in the comparison group. Fasting insulin levels were elevated in 4 propositi and 6 of the comparison group; mean insulin levels were within the normal range for both groups. In the comparison of lipids for the subset of patients before and after HAART therapy, HAART was associated with significant increases in total cholesterol, apolipoprotein B, and HDL cholesterol. Changes in body habitus caused by redistribution of fat occur commonly in women receiving HAART. Serum lipid abnormalities also are common during HAART and appear to be as frequent in women who do not experience clinically apparent body fat redistribution as in those who do. The observed changes in body fat distribution and in serum lipid levels are alterations that have been strongly correlated with increased risk for cardiovascular disease. Therefore, an understanding of the basis of these phenomena, and the risks with which they may be associated in this population, will be important for therapeutic decision making in women with HIV disease.

Low concentrations of high-density lipoprotein cholesterol (HDL-C) are a recognized risk factor for atherosclerotic cardiovascular disease. Exercise is often recommended to increase HDL-C, but the effect of exercise training on HDL levels and metabolism in subjects with low HDL concentrations is not well defined. The present study compared the HDL response to 12 months of supervised endurance exercise training without weight loss in 17 men aged 26 49 years with initially low ( < 40 mg/dl, N=7) or normal ( > 44 mg/dl, N=10) HDL-C levels. HDL-C levels and HDL apolipoprotein metabolism were assessed while the subjects consumed controlled diets before and after the year of training. Increases in total (5.1+/-2.8 versus 1.9+/-4.2 mg/dl, P=0.08) and HDL2 (3.8+/-2.9 versus 0.4+/-1.1 mg/dl, P=0.01) cholesterol were greater in men with normal initial HDL-C levels. Catabolic rates for HDL apolipoproteins decreased 7-14% and biological half-lives increased 10-15% after exercise training in subjects with normal HDL, but were unchanged in the low HDL-C group. HDL apolipoprotein synthetic rates were not consistently affected by exercise training in either group. Postheparin lipoprotein lipase activity increased 27%, the clearance rate of intravenous triglycerides increased 14%, and apolipoprotein B levels decreased 16% with training in subjects with normal HDL-C but were unchanged in the low HDL-C group. We conclude that the ability to increase HDL-C levels through endurance exercise training is limited in subjects with low initial HDL-C, possibly because exercise training in such subjects fails to alter triglyceride metabolism.

Purpose: Although public health interventions have not specifically targeted high density lipoprotein (HDL) cholesterol, observed changes in the prevalence of other cardiovascular risk factors would be expected to have differential effects on HDL. This study examined secular trends in HDL in relation to changes in other cardiovascular risk factors for the years 1981 through 1993 in the Pawtucket Heart Health Program (PHHP) study communities., Methods: Nonfasting HDL levels were assessed in 12,223 respondents to six biennial population random sample surveys., Results: Between 1981 and 1993, mean HDL cholesterol declined by 0.08 mmol/L in both men and women after adjustment for age, city, education, hormone use, medications, recent alcohol use, smoking, regular exercise, body mass index (BMI), and total cholesterol, (p for trend < 0.001). There was no apparent laboratory explanation for the trend which occurred concurrent with decreased smoking prevalence, increasing BMI and decreased prevalence of recent alcohol use. Decreasing HDL cholesterol was observed consistently across subgroups defined by smoking, alcohol use and BMI., Conclusions: Although several favorable cardiovascular risk factor trends have been observed in recent decades, declining HDL cholesterol is also of interest, particularly in conjunction with population increases in BMI.

We have identified a kindred in Providence, RI, deficient in hepatic triglyceride lipase (HL). The two affected brothers have coronary heart disease and elevated levels of triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, and apolipoprotein [apo] A-I. The lipoprotein lipase (LPL) activity is normal. We and others have postulated that the effects of oral anabolic steroids on HDL metabolism are mediated by HL. To test this hypothesis, we treated these two men and two controls with the oral androgen stanozolol (6 mg/d) for 2 weeks. Consistent with other reports, HL activity increased a mean of 277% in controls with a concomitant decrease in HDL cholesterol (49%), HDL2 cholesterol (90%), HDL3 cholesterol (16%), and apo A-I (41%) and no change in apo A-II. Although stanozolol failed to induce HL activity in the HL-deficient man, HDL cholesterol, HDL2 cholesterol, and apo A-I were reduced a mean of 20%, 48%, and 32%, respectively. In contrast to controls, HDL3 cholesterol (46%) and apo A-II (14%) increased in HL-deficient subjects. Stanozolol treatment also increased LPL activity (124% +/- 86%, n = 4) and decreased lipoprotein(a) ([Lp(a)] 66% +/- 3%, n = 3) in the three men with detectable levels. The data indicate that in addition to stimulation of HL activity, stanozolol treatment changes HDL cholesterol concentration and subfraction distribution by other mechanisms.

Elevated total homocysteine (tHcy) levels are associated with increased risk for atherosclerotic cardiovascular disease. tHcy levels are higher in men than in women, and estrogen replacement therapy may reduce tHcy levels in postmenopausal women. The effect of androgenic hormones on tHcy levels in men has not been examined. The present study determined the effect of supraphysiologic doses of testosterone, with or without its aromatization to estradiol, on fasting tHcy levels in 14 normal male weightlifters aged 19-42 years. Subjects received testosterone-enanthate (200 mg/week intramuscularly), the aromatase inhibitor, testolactone (1 g/day orally), or both drugs together in a crossover design. Each treatment lasted 3 weeks and each treatment was separated by a 4-week washout. Both testosterone regimens increased serum testosterone levels, whereas estradiol increased only during testosterone alone. Mean tHcy levels were not significantly altered when testosterone was given alone or together with testolactone. Testolactone did not significantly influence tHcy levels. We conclude that short-term, high-dose testosterone administration does not affect fasting tHcy levels in normal men.

We administered testosterone, with or without the aromatase inhibitor testolactone, to determine the effects of testosterone and its aromatization to estradiol on Lp(a) levels in normal men. Average Lp (a) values decreased by 37% during testosterone alone and by 28% when testosterone and testolactone were combined, suggesting that testosterone reduces Lp(a) in men primarily by an androgenic effect and not by its conversion to estradiol.

We compared the effects of freezing serum on the determination of high-density lipoprotein (HDL) subfractions by two dual-precipitation methods, heparin and manganese chloride/dextran sulfate (HM/DS) (Gidez et al., J Lipid Res 1982;23:1206-23) and DS/DS (Warnick et al., Clin Chem 1982;28:1574), and by ultracentrifugation. Storing serum for 1 month at -70 degrees C resulted in reduced HDL3-cholesterol by ultracentrifugation and reduced total and HDL3-cholesterol by the DS/DS method. There was no change in either total HDL-cholesterol or HDL3-cholesterol with the HM/DS method. Additional studies involving only HM/DS indicated that total HDL-cholesterol in serum stored at 4 degrees C begins to decline after 3 days (-3.1 +/- 3.5%, P < 0.1). HDL was stable at -20 degrees C for 2 weeks but both total and HDL3-cholesterol decreased significantly after 1 month. Storage of serum at -70 degrees C resulted in no changes for 1 year; however, at 18 months, HDL3-cholesterol was reduced 13% (P = 0.002). We conclude that HDL subfractions can be determined accurately in serum as well as in plasma after storage at -70 degrees C for up to 1 year.

Serum amyloid A (SAA) is an acute phase protein that in the blood is bound to high density lipoproteins; SAA is secreted mainly by hepatocytes, and its concentration increases in the blood up to 1000 times during an inflammatory response. At present, its biological function is unclear. Since some forms of secondary amyloidosis are caused by deposition in tissues of peptides derived from the SAA and leukocytes seem to be involved in this process, we investigated the effect of human SAA on human monocytes and polymorphonuclear cells (PMN). When recombinant human SAA (rSAA) was used at concentrations corresponding to those found during the acute phase (> 0.8 microM), it induced directional migration of monocytes and polymorphonuclear leukocytes. Preincubation of rSAA with high density lipoproteins blocked this chemoattractant activity for both monocytes and PMN. rSAA also regulated the expression of the adhesion proteins CD11b and leukocyte cell adhesion molecule 1 and induced the adhesion of PMN and monocytes to umbilical cord vein endothelial cell monolayers. When subcutaneously injected into mice, rSAA recruited PMN and monocytes at the injection site. On the basis of these data, we suggest that SAA may participate in enhancing the migration of monocytes and PMN to inflamed tissues during an acute phase response.

Numerous studies have examined factors regulating high-density lipoprotein cholesterol (HDL-C) levels in male endurance athletes, but few studies have examined HDL-C regulation in female athletes. The present study compared lipid and lipoprotein concentrations, postheparin lipolytic activities, and the clearance rate (K2) of triglycerides following an intravenous fat infusion in 12 female distance runners (aged 33 +/- 9 years, mean +/- SD) and 13 sedentary women (33 +/- 9 years). Runners were leaner and had greater maximum oxygen uptake values than controls. Runners also had nonsignificantly lower triglyceride (53 +/- 15 v 65 +/- 13 mg/dL) and higher HDL-C (62 +/- 14 v 52 +/- 8 mg/dL, P = .06). Lipoprotein lipase activity (LPLA) was 33% greater (P < .05) and fat clearance (K2) was 27% faster (P < .01) in the trained women, and LPLA correlated directly with K2 (r = .61) and HDL-C (r = .62) in this group (P < .05 for both). K2 was directly related to HDL-C in the athletes (r = .57, P = .06), and also when the active and sedentary women were combined (r = .43, P < .05). These results suggest that increased LPLA and enhanced plasma triglyceride clearance may contribute to the HDL-C levels of physically active premenopausal women.

Stanozolol, an oral 17 alpha-alkylated androgen, increases hepatic triglyceride lipase activity (HTGLA) and decreases high-density lipoprotein cholesterol (HDL-C) levels, whereas intramuscular testosterone has comparatively little effect. In the present study, we tested the hypothesis that aromatization of androgen to estrogen blunts the lipid and lipase effects of exogenous testosterone. Fourteen male weightlifters received testosterone enanthate (200 mg/wk intramuscularly), the aromatase inhibitor testolactone (250 mg four times per day), or both drugs together in a randomized cross-over design. Serum testosterone level increased during all three drug treatments, whereas estradiol level increased only with testosterone alone (+47%, P < .05), demonstrating that testolactone effectively inhibited testosterone aromatization. Testosterone decreased HDL-C(-16%, P < .05), HDL2-C(-23%, NS), and apoprotein (apo) A-I (-12%, P < .05) levels, effects that were consistently but not significantly greater with simultaneous testosterone and testolactone administration (HDL-C, -20%; HDL2-C, -30%; apo A-I, -15%; P < .05 for all). In contrast, both testosterone regimens decreased HDL3-C levels by 13% (P < .05 for both). HTGLA increased 21% during testosterone treatment and 38% during combined testosterone and testolactone treatment (P < .01 for both). Lipoprotein lipase activity (LPLA) increased only during combined testosterone and testolactone treatment (+31%, P < .01), suggesting that estrogen production may counteract the effects of testosterone on LPLA. Testolactone alone had little effect on any lipid, lipoprotein, apoprotein, or lipase concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of serum amyloid A (SAA) was studied in isolated perfused livers of mice treated with either a single injection of casein to induce an acute phase response or with 14 daily casein injections to maintain chronic inflammation. Littermates administered sterile saline served as controls. Radioiodinated SAA and apolipoprotein A-I, reconstituted with high-density lipoproteins in vivo, were studied in parallel. Degradation was monitored by appearance of acid-soluble radioactivity in the perfusate. Induction of an acute phase response reduced hepatic catabolism of SAA by 14% (from 8.6 +/- 1.2% to 7.4 +/- 1.1%/g liver in 3 hr, P less than 0.05, n = 16). The acute phase response had no effect on apolipoprotein A-I degradation or bile production. Livers from animals receiving 14 daily injections of casein were 31% less active than control livers at degrading SAA (8.1 +/- 1.6%/g/3 hr for treated group vs. 11.7 +/- 2.3%/g/3 hr for control group, P less than 0.025). Apolipoprotein A-I degradation was decreased but differences were not statistically significant and bile production was the same in both treatment groups. However, livers from treated animals were larger (mean weight 1.8 g) than those from controls (1.5 g) (P less than 0.05), although amyloid fibrils were not detected by Congo red stain. The size of the degradation products was analyzed by column chromatography. Elution profiles of perfusates from livers of chronically inflamed animals contained a peak corresponding to the molecular weight of amyloid A which was not present in perfusates from control liver. We conclude that hepatic catabolism of SAA is decreased both early and late in an inflammatory response and intermediate degradation products corresponding in size to amyloid A are released into the circulation following prolonged inflammation.

We have investigated the protease activity, present in human serum, that digests the serum amyloid A (SAA) protein. SAA radiolabeled with 125I was incubated at 37 degrees C with serum and plasma and analyzed for degradation products by alkaline urea-polyacrylamide gel electrophoresis and gel filtration chromatography. Serum initially digested SAA to intermediates of 3000-5000 in molecular weight, and these were further degraded to smaller peptides with prolonged incubation. SAA was not degraded by plasma anticoagulated with ethylenediaminetetraacetic acid (EDTA) or heparin. Recalcification of plasma anticoagulated with EDTA led to the generation of protease activity against SAA whereas EDTA plasma defibrinated with thrombin was inactive. We employed both nonselective and selective protease inhibitors and synthetic substrates for kallikrein and plasmin to further characterize the serum protease. These studies demonstrated that degradation of SAA is not directly attributable to enzymes involved in coagulation, kinin formation, or fibrinolysis, but the unidentified protease may be activated by one of the clotting factors. The specificity of the SAA degradation was demonstrated in experiments with three of the well-characterized apolipoproteins. Apolipoproteins A-I, C-I, and C-III-1, which also associate with the plasma high-density lipoproteins, were not degraded by serum although they were good substrates for purified thrombin and plasmin.

Serum amyloid A and high density lipoprotein (HDL) interrelationships were evaluated in 11 normal men during an acute phase response induced by the inflammatory steroid etiocholanolone. Compared with baseline, HDL-cholesterol levels were significantly elevated at 30 h but not at 50 h (P less than 0.05) after etiocholanolone. A-apoprotein concentrations were unchanged at 30 h but were reduced at 54 h (P less than 0.01). Four subjects were sampled every 6-8 h for 5 days. Two men had peak SAA concentrations of 30 and 33 mg dl-1. Their A-apoprotein levels declined as SAA rose and remained low even after SAA levels had returned to baseline. High density lipoprotein cholesterol levels did not fall, however, when SAA was increasing, and fell only after SAA levels declined. No changes in HDL-cholesterol or protein were observed in two subjects whose peak SAA concentrations were 10 and 12 mg dl-1. These observations suggest that a threshold level of acute phase response is required before HDL reductions occur. Column chromatography of SAA-rich plasma did not demonstrate the presence of either SAA or A-apoproteins that were unassociated with lipoproteins. Serum amyloid A, moreover, demonstrated little capacity to displace A-proteins from HDL at SAA concentrations typically observed during the acute phase response. We infer from these studies that SAA may substitute for the A-apoproteins and temporarily maintain HDL-cholesterol levels; but that low HDL levels during the acute phase response are likely due to reduced A-protein synthesis rather than displacement by SAA.

Degradation of serum amyloid A (SAA) was studied in the isolated perfused rat liver. Radioiodinated SAA was reconstituted with high density lipoproteins (HDL) and administered to rats. Plasma was taken 1 h later, and the HDL were isolated for use as tracer. HDL-bound 125I-SAA was cleared from the plasma of intact animals at a rate similar to SAA in native human HDL. Catabolism of SAA and HDL apoproteins was studied in parallel in the perfused liver. In a 3-h perfusion, 21% of SAA was degraded in contrast to 13% of apoC-III, 7% of apoA-I, and 6% of apoA-II. SAA1 (47% in 3 h) was degraded more rapidly than SAA5 (37%) although their in vivo clearance rates were similar. Degradation of SAA was inhibited when lipoproteins were added to the perfusate. At a protein concentration of 0.15 mg/ml, low density lipoproteins inhibited 47%, HDL 62%, and SAA-rich HDL 75%. Lipid-free normal HDL (0.3 mg/ml perfusate) did not appreciably affect SAA degradation; however, delipidated SAA-rich HDL (0.3 mg of protein/ml; 0.02 mg of SAA/ml) inhibited SAA degradation by 40%. Isolated perfused mouse liver proved more effective than rat liver in degrading SAA (5.3% versus 2.8%/g of liver/h). Degradation appeared to be mediated by cell-associated enzymes since perfusate, which had been recirculated through the liver for 3 h, accounted for less than 15% of the total degradation. Partial (38%) hepatectomy did not significantly reduce apoA-I clearance but reduced that of SAA by 16%, providing additional evidence for hepatic SAA catabolism. We conclude from these studies that SAA is catabolized independently of other HDL proteins, that association with lipoproteins retards SAA clearance, and that SAA catabolism is, in part, a specific process.

We recently demonstrated that the serum amyloid A proteins (SAA) occur in six related polymorphic forms of indistinguishable molecular weights and COOH-terminal sequence. We have now obtained very homogeneous preparations of four of these proteins and shown that their amino acid compositions are similar but not identical. Two of these, SAA1 and SAA4, have the same 20 NH2-terminal residues despite striking differences in electrophoretic mobility and solution properties. SAA5 and SAA2, respectively, lack one and three of the NH2-terminal residues common to SAA1 and SAA4. The data are consistent with the postulate that some of the SAA polymorphs are products of different genes.

Serum amyloid A proteins (SAA), presumed precursors of the tissue amyloid A proteins (AA) characteristic of secondary amyloidosis, have been isolated from the plasma high-density lipoproteins (HDL) of normals after etiocholanolone-induced inflammation and from patients with Wegener's granulomatosis, systemic lupus erythematosis, juvenile rheumatoid arthritis, Waldenström's macroglobulinemia, and Goodpasture's syndrome. At least six polymorphic forms of SAA wer identified among the low molecular weight proteins of HDL, and these comprosed up to 27% of the total HDL protein. Gel and ion-exchange chromatography permitted isolation of the SAA polymorphs in homogeneous form. Their amino acid compositions were very similar, they were indistinguishable in cationic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems, and each had the terminal sequency COOH-Tyr-Lys-Phe-. Charge heterogeneity in anionic-urea polyacrylamide gel electropherograms was unaffected by neuaminidase treatment, and none of the SAA protein bands stained with the periodate-Schiff reagent. The two major SAA polymorphs, designated SAA4 and SAA5 according to their order of elution from DEAE-cellulose, had different NH2-terminal sequences. Manual Edman degradation demonstrated NH2-arg-ser-phe-phe- for SAA4 and NH2-ser-phe-phe- for SAA5. This NH2-terminal heterogeneity corresponds to that most frequently reported for AA and suggests that microheterogeneity in SAA may underlie that already documented in AA. Sufficient quantitites of the other SAA polymorphs were not available for similar analyses, but the amino acid compositions do not indicate that NH2-terminal heterogeneity accounts for all of the observed polymorphism. Artifactual polymorphism also appears unlikely, and the heterogeneiy of SAA may reflect origin from more than one cell type with or without posttranslational modificaton. We calculate from quantitative COOH-terminal analyses that SAA is of 11,000-11,900 mol wt. Primary structure studies have shown AA t be a single chain protein of 76 residues, and SAA, therefore, appears to contain a peptide of 33 amino acids that is missing from AA.

Plasma concentrations of serum amyloid A (SAA), high density lipoprotein (HDL) cholesterol, non-HDL cholesterol, and apolipoproteins (Apo) A-I and B were measured daily for 6 days in 10 patients following myocardial infarction (MI) and in 10 secular controls admitted to a coronary care unit. SAA concentrations peaked 3 days following MI (mean 47 mg/dl) and correlated with creatine kinase (CK) (r = 0.67, P less than 0.001). Non-HDL cholesterol and Apo B fell 15 and 18%, respectively, reached nadirs 3-4 days after MI and were inversely related to CK concentrations (P less than 0.01 for both). HDL cholesterol levels, in contrast, increased 15% and were significantly higher than baseline by day 3 when SAA concentrations were maximum. HDL cholesterol subsequently fell in parallel with SAA and had returned to baseline by day 6. Apo A-I declined throughout the 6 days of observation and was 13% lower than initial values on day 6 (P less than 0.05). The Apo A-I reduction was inversely related to both CK and SAA concentrations. There were no significant changes in any of the analytes in control subjects. We conclude that Apo A-I and possibly Apo B containing lipoproteins are negative acute phase reactants. HDL cholesterol is transiently elevated after MI despite decreasing Apo A-I levels and this may relate to incorporation of SAA into HDL particles.

Continued studies of the macrophage-derived mediator of SAA synthesis (SAA Stimulating Factor) confirm our previous observations that SAASF copurified with leukocytic pyrogen (LP) and lymphocyte activating factor (LAF). Moreover, new data demonstrate three separate isoelectric points for human LP-LAF-SAASF each of which possess the three biological activities. During the purification of 15,000 MW LP from crude stimulated mononuclear cell supernatants, only those fractions with pyrogenic activity in rabbits caused augmented stimulation of lymphocytes (LAF) and induced SAA synthesis in mice. Purified human LP stimulated isolated mouse hepatocytes in vitro to synthesize SAA in a dose-responsive manner. Colchicine treatment of hepatocytes led to decreased secretion of SAA into the medium and to an intracellular accumulation of SAA. Messenger RNA was isolated from the livers of endotoxin-stimulated mice and translated in a wheat-germ cell-free system. A major product was identified at 13-14,000 MW. Immunoprecipitation with anti-mouse AA identified several bands on autoradiography of polyacrylamide gels. These larger SAA precursors may account for the previously noted heterogeneity of human SAA, comprising at least 6 SAA isomers, of similar molecular weight but different solubility and electrophoretic charge characteristics. Two monoclonal antibodies (IgM-K and IgG1-K) have been prepared using standard cell hybridization techniques. They are directed at the variable COOH terminal region of SAA since they detect differences between the 6 human SAAs but do not react with human, monkey, dog or mouse AA proteins, human AP, C-reactive protein, IgG nor albumin. These antibodies will be useful in examining the origin, structure and function of SAA.

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.

The interaction of synthetic dimyristoyl phosphatidylcholine (lecithin) liposomes with isolated apoC-I and apoC-III proteins from very low density lipoproteins has been studied by microcalorimetry. Complex formation is a highly exothermal process characterized by a maximal enthalpy of -130 kcal/mol (-544 kJ) apoC-III-1 and -65 kcal/mol apoC-I proteins (-272 kJ). The complex composition determined after its isolation by ultracentrifugal flotation agrees with the value derived from the enthalpy binding curves. The binding of a constant amount of dimyristoyl lecithin to apoprotein mixtures containing various proportions of apoA-I and apoC-III failed to demonstrate the existence of any preferential association between the two apoproteins, in contrast with results obtained previously with apoA-I/apoA-II protein mixtures. Finally the various contributions to the enthalpy of binding such as that arising from an increase in apoprotein helicity have been evaluated. A classification of the apolipoproteins according to their lipid-binding affinity is proposed as: apoA-II congruent to apoC-III greater than apoC-I greater than apoA-I proteins.

We used antisera to human A and C apolipoproteins to identify homologues of these proteins among the high-density lipoprotein apoproteins of Macaca fascicularis (cynomolgus) monkeys, and NH2-terminal analysis was used to verify the homology. The NH2-terminal sequence of the M. fascicularis apoA-I is identical with that of another Old World species, Erythrocebus patas, and differs from human apoA-I at only 4 of the first 24 residues. M. fascicularis apoA-II contains a serine for cysteine replacement at position 6 and is therefore monomeric like the apoA-II from all species below apes. Human and monkey apoA-II are not otherwise different through their first 25 residues. About 20% of M. fascicularis apoC-I aligns with human apoC-I through residue 22, and 80% lacks an NH2-terminal dipeptide. Otherwise, the monkey apoC-I differs from the human protein at only 2 of 25 positions. Two forms of M. fascicularis apoC-II were identified. ApoC-II1 is highly homologous with human apoC-II, whereas an NH2-terminal hexapeptide is absent from apoC-II2. ApoC-II2 was the predominant species, and apoC-II1 appears to represent a propeptide from which a hexapeptide prosegment is cleaved at a Gln-Asp bond. Both forms of monkey apoC-II are potent activators of lipoprotein lipase. There are two polymorphic forms of M. fascicularis apoC-III, and their electrophoretic mobilities become identical after treatment with neuraminidase. Except for a glycine for serine substitution at position 10, the first 15 NH2-terminal residues of M. fascicularis and human apoC-III are the same.

The serum amyloid A proteins (SAA) occur in plasma in six polymorphic forms that are associated with the high-density lipoproteins (HDL). We studied two of the SAA proteins, SAA1 and SAA4, which have the same amino- and carboxy-terminal residues but different solution properties and electrophoretic mobilities, to determine whether they are interconverted in plasma in vivo. They were radioiodinated in vitro, incorporated into HDL, and administered to cynomolgus monkeys. Both remained associated with HDL for at least 6 h, had similar plasma die-away curves, and retained their characteristic electrophoretic mobilities, suggesting they are not related as precursor and product. The plasma clearance of the most prevalent SAA species, SAA4, was also simultaneously compared with the human A-I and C-III-2 apolipoproteins. Human apolipoprotein A-I decayed from plasma at a rate comparable to that of monkey HDL proteins. Apolipoprotein C-III-2 was cleared more rapidly and SAA4 at an even greater rate. These findings suggest that SAA are either dissociated from HDL before clearance from plasma or that SAA are contained in an HDL subspecies with metabolic fate different from that of most HDL particles.