Effects of tamoxifen on cardiovascular risk factors in postmenopausal women after 5 years of treatment.

BACKGROUND: Adjuvant tamoxifen therapy for breast cancer has been given for a period of several years. Cardiovascular diseases increased in incidence rapidly in women older than 60 years. Favorable changes in cardiovascular risk factors have been seen with 2 years of tamoxifen therapy, and lower rates of myocardial infarction and of hospitalization for heart disease have been observed in tamoxifen-treated women. PURPOSE: We sought to evaluate changes in risk factors for cardiovascular diseases in postmenopausal women after therapy with tamoxifen for 5 years. METHODS: Five years after their initial entry in a 2-year randomized, placebo-controlled toxicity study, we re-examined 62 of the original 140 disease-free, axillary node-negative postmenopausal breast cancer patients. These 62 patients were women available for study because they had not suffered major illness and had continued on either the tamoxifen or no-tamoxifen regimen to which they had been originally randomly assigned for the entire 5 years. Patient and control blood samples were analyzed for total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and subfractions, triglycerides, apolipoprotein AI, apolipoprotein B, lipoprotein(a), fibrinogen, glucose, and platelets. RESULTS: At base line for all measurements except atherogenic lipoprotein [lipoprotein(a)], the 30 long-term tamoxifen recipients and the 32 long-term no-tamoxifen recipients were not significantly different. After 5 years, levels of total serum cholesterol (P < .001), LDL cholesterol (P < .001), and lipoprotein(a) (P = .001) were significantly lower, and apolipoprotein AI levels were significantly higher (P < .001) in the tamoxifen-treated group compared with the no-tamoxifen group. Apolipoprotein B levels increased to a greater extent in the no-tamoxifen than in the tamoxifen group (P < .001). After 5 years, fibrinogen level decrease and triglyceride level increases in the tamoxifen group compared with the no-tamoxifen group were of borderline statistical significance and HDL cholesterol levels were not different in the two groups. CONCLUSION: Favorable changes in lipid, lipoprotein, and fibrinogen levels seen early in tamoxifen therapy in postmenopausal women persist with treatment of 5 years. IMPLICATIONS: The types and magnitude of changes in cardiovascular risk factors seen here with tamoxifen are similar to a certain extent with those seen with estrogen supplements. Further risk-factor and ethnic-group data are needed to estimate the magnitude of expected benefits of tamoxifen treatment on incidence of heart disease.

Effects of tamoxifen therapy on lipid and lipoprotein levels in postmenopausal patients with node-negative breast cancer.

We conducted a 2-year, randomized, double-blind, placebo-controlled toxicity trial of therapy with tamoxifen (10 mg twice a day) in 140 postmenopausal women with a history of breast cancer and histologically negative axillary lymph nodes. These women had been treated with surgery with or without radiotherapy. At a 3-month evaluation, tamoxifen-treated women showed a significant decrease in fasting plasma levels of total cholesterol and low-density lipoprotein (LDL) cholesterol, which persisted at 6- and 12-month evaluations. During the first 12 months, plasma triglyceride levels increased; small but significant decreases in high-density lipoprotein cholesterol (HDL) were observed in tamoxifen-treated women, but ratios of total cholesterol to HDL cholesterol and of LDL to HDL cholesterol changed favorably. While data relating lipid/lipoprotein profiles and cardiovascular disease are limited in women, current evidence suggests that total cholesterol and possibly low-density lipoprotein cholesterol are risk factors. We conclude that during the first 12 months of treatment, tamoxifen exerts a favorable effect on the lipid profile in postmenopausal women with early stage breast cancer.

Fatty acid utilization by L1210 murine leukemia cells.

L1210 murine leukemia cells grow in an ascites plasma that contains lipids, including 0.62 +/- 0.046 (S.E.) MICRONEq free fatty acid per ml. in vitro incubations demonstrated that isolated L1210 cells readily utilize free fatty acid that is added to the incubation medium. When the cells were incubated with albumin-bound [1-14C]palmitate, about 12 times more radioactivity was incorporated into cell lipids than was oxidized to CO2. Triacylglycerols contained 1.5 to 4 times more radioactivity than phospholipids, and from 48 to 69% of the phospholipid radioactivity was recovered in the choline phosphoglycerides. [1-14C]Palmitate utilization increased as the fatty acid concentration of the medium was raised, the largest increase occurring in the triacylglycerol fraction. Palmitate utilization also was increased by the presence of carbohydrates in the medium, their effectiveness (in descending order) being glucose, mannose, galactose, fructose, and glycerol. By contrast, ribose did not produce any stimulatory effect. During a 1-hr incubation, between 82 and 87% of the [1-14C]palmitate that was taken up remained as palmitic acid. From 8 to 15% was elongated to stearate, and only 2 to 3% was desaturated to palmitoleate and oleate. Based upon the lipid content, growth rate, and palmitate utilization rate of the cells, it appears that a major portion of the lipid requirements of the L1210 cell may be supplied by the fatty acid contained in the ascites plasma. In addition, our results suggest that most of the saturated fatty acid taken up is incorporated into cell lipids without structural modification.

Purple grape juice improves endothelial function and reduces the susceptibility of LDL cholesterol to oxidation in patients with coronary artery disease.

BACKGROUND: In vitro, the flavonoid components of red wine and purple grape juice are powerful antioxidants that induce endothelium-dependent vasodilation of vascular rings derived from rat aortas and human coronary arteries. Although improved endothelial function and inhibition of LDL oxidation may be potential mechanisms by which red wine and flavonoids reduce cardiovascular risk, the in vivo effects of grape products on endothelial function and LDL oxidation have not been investigated. This study assessed the effects of ingesting purple grape juice on endothelial function and LDL susceptibility to oxidation in patients with coronary artery disease (CAD). METHODS AND RESULTS: Fifteen adults with angiographically documented CAD ingested 7.7+/-1.2 mL. kg(-1). d(-1) of purple grape juice for 14 days. Flow-mediated vasodilation (FMD) was measured using high-resolution brachial artery ultrasonography. Susceptibility of LDL particles to oxidation was determined from the rate of conjugated diene formation after exposure to copper chloride. At baseline, FMD was impaired (2.2+/-2. 9%). After ingestion of grape juice, FMD increased to 6.4+/-4.7% (P=0.003). In a linear regression model that included age, artery diameter, lipid values, and use of lipid-lowering and antioxidant therapies, the effect of grape juice on FMD remained significant (mean change 4.2+/-4.4%, P<0.001). After ingestion of grape juice, lag time increased by 34.5% (P=0.015). CONCLUSIONS: Short-term ingestion of purple grape juice improves FMD and reduces LDL susceptibility to oxidation in CAD patients. Improved endothelium-dependent vasodilation and prevention of LDL oxidation are potential mechanisms by which flavonoids in purple grape products may prevent cardiovascular events, independent of alcohol content.

Use of human immunodeficiency virus-1 protease inhibitors is associated with atherogenic lipoprotein changes and endothelial dysfunction.

BACKGROUND: Human immunodeficiency virus protease inhibitors (HIV PIs) are associated with hyperlipidemia, hyperglycemia, and obesity; however, it is not known whether they increase risk of atherosclerotic vascular disease. The purposes of this study were to characterize the lipoprotein abnormalities associated with use of HIV PIs in individuals with HIV infection and to determine the pathophysiological significance of these changes by assessing their effect on endothelial dysfunction. METHODS AND RESULTS: This was a cross-sectional study of 37 adults with HIV-1 infection who were receiving antiretroviral therapy. Twenty-two were taking HIV PIs (group 1); 15 were not (group 2). Lipids and lipoproteins were measured by enzymatic techniques and nuclear magnetic resonance spectroscopic analysis. Flow-mediated vasodilation (FMD) of the brachial artery was measured by high-resolution ultrasound. Subjects in both groups were similar in regard to age, time since diagnosis of HIV infection, and CD4 cell count. Group 1 subjects had higher total cholesterol (5.68 versus 4.42 mmol/L, P=0.007) and triglyceride (4.43 versus 1.98 mmol/L, P=0.009) levels, characterized by elevated levels of IDL and VLDL. Subjects in group 1 had impaired FMD (2.6+/-4.6%), indicative of significant endothelial dysfunction. Group 2 subjects had normal FMD (8.1+/-6.7%, P=0.005). In group 1, chylomicron, VLDL, IDL, and HDL cholesterol levels predicted FMD. CONCLUSIONS: Use of HIV PIs is associated with atherogenic lipoprotein changes and endothelial dysfunction. Because these metabolic and vascular changes predispose to atherosclerosis, monitoring and treatment of dyslipidemia in patients taking these medications is warranted.

Severe hypercholesterolemia mediated by lipoprotein X in patients with chronic graft-versus-host disease of the liver.

We describe a series of cases of extreme hypercholesterolemia mediated by lipoprotein X in patients with chronic graft-versus-host disease of the liver after an allogeneic bone marrow transplant. All of the patients presented with a total cholesterol in excess of 1000 mg/dl (25.9 mmol/l). At the time they were also noted to have pseudohyponatremia. Cholesterol appeared to be predominantly carried by lipoprotein X. Intrahepatic cholestasis leading to reflux of bile lipoproteins into the bloodstream and subsequent formation of protein X appears to be the mechanism underlying this phenomenon. Complications, including retinal cholesterol thromboembolism and cholesteroloma of the lung have been seen in the patient with the highest cholesterol levels. Severe hypercholesterolemia is an important, and likely more common than previously reported, long-term complication of allogeneic hematopoietic stem cell transplantation. It is important for clinicians to familiarize themselves with the diagnostic and therapeutic challenges this condition presents.

Lipid testing for the year 2000 and beyond.

Lipid testing has progressed from early measurements of total lipid by extraction and weighing to assess the fat content of the specimen. This nonspecific approach to lipid testing has been replaced in clinical laboratories by automated and quantitative procedures that avoid the extraction process. Instead, selective enzymes are utilized in reaction schemes to quantitate the individual lipid classes present in patient specimens. For example, cholesterol esterase and oxidase are used on a routine basis to measure total cholesterol in plasma and serum specimens. Similar use of other enzyme systems has permitted triglycerides and phospholipids to be measured by clinical laboratories. Lipid and lipoprotein measurements have advanced considerably from the early nonspecific extraction and gravimetric analysis schemes to the specific automated procedures that are commonly used today. However, as lipids and lipoproteins increased in their clinical usefulness as cardiovascular risk assessment tools, the search intensified for newer approaches to measure these entities more easily and more accurately. The influence of National Cholesterol Education Program has played a key role in highlighting the importance of lipids and lipoprotein analysis. Today, lipid testing is available outside the traditional laboratory environments - drugstores sell units that individuals can use at home to assess cholesterol levels. Lipid testing has come a long way, and we have only begun to experience some of the remarkable changes for the future.

Plasma lipoprotein changes in humans induced by beta-interferon.

Two groups of patients were administered either 4.5 X 10(6) U or 90 X 10(6) U each of recombinant DNA-derived interferon-beta serine (IFN-beta ser) i.v. daily for 10 days. IFN-beta ser affected lipoprotein lipids of patients in a dose dependent fashion. A decrease in plasma total cholesterol concentration occurred 24 h after therapy was initiated, regardless of dose. A dose-related decrease in plasma cholesterol concentration of 9% and 23% for patients on the low dose and high dose respectively occurred after 9 days of therapy. The plasma total cholesterol concentration decrease resulted primarily from a decrease in LDL cholesterol of 28% and 50% for patients on low and high doses respectively of IFN-beta ser. HDL-cholesterol was not significantly affected by IFN-beta ser administration. A dose-related increase in plasma triglyceride concentration occurred during IFN-beta ser, increasing 74% for patients on low dose and 136% for patients on high doses. This increase was only observed after 9 days on IFN-beta ser. Cholesterol reduction and triglyceride increases followed different time courses indicating different mechanisms may be involved.

Triglyceride and cholesterol concentrations in whole serum and in serum lipoproteins in Reye syndrome.

Triglyceride and cholesterol concentrations in whole serum and in the serum lipoprotein fractions were measured during the course of hospitalization in six patients with Reye syndrome, four of whom survived. Very low density lipoprotein (VLDL) triglycerides were significantly lower on admission than on the last day of hospitalization. However, no VLDL triglyceride value was below the normal range. Triglyceride transport was increased in low density lipoprotein (LDL) and high density lipoprotein (HDL) fractions. LDL and HDL cholesterol concentrations were low on admission and decreased further during hospitalization. The changes in LDL and HDL cholesterol concentrations were more severe among nonsurvivors. No HDL cholesterol was detected in nonsurvivors on the last day of hospitalization. These results suggest that decreased VLDL triglycerides may not play an important role in the development of fatty liver and that decreased LDL and HDL cholesterol concentrations may be of prognostic value in Reye syndrome.

Synergistic and antagonistic effects of combinations of cyclosporine A and its metabolites on inhibition of phytohemagglutinin-induced lymphocyte transformation in vitro.

Cyclosporine A (CsA) and purified CsA metabolites were tested alone and in combination in cell culture to determine their effects on phytohemagglutinin (PHA)-induced lymphocyte proliferation. CsA was significantly more inhibitory than its metabolites at all concentrations tested (0-1000 ng/mL). CsA exerted maximum inhibition (70% decrease in [methyl-3H]thymidine incorporation) at concentrations of 300 ng/mL or greater; metabolites M1, M17, and M21 depressed the response 46, 39, and 23%, respectively, at 300 ng/mL. Metabolites M8, M18, M26, M25, M13, and M203-218 were non-inhibitory. When combinations of M17 and CsA were tested for the effects on PHA-induced lymphocyte transformation, a synergistic effect occurred at combinations of low concentrations of M17 and CsA and an antagonistic effect at the higher concentrations. Of the 49 combinations of CsA and M17 tested, 30 were antagonistic, 16 synergistic and 3 undecided (approaching addition). When 49 combinations of CsA and the non-immunosuppressive metabolite M8 were tested, 29 of the 49 combinations were synergistic, 17 antagonistic, 1 additive and 2 undecided (approaching addition). Of the 29 synergistic combinations, 14 were strongly synergistic. The importance of the interaction of CsA and metabolites to the immunopharmacology of CsA therapy is discussed.

Ophthalmic observations in lecithin cholesterol acyltransferase deficiency.

Lecithin cholesterol acyltransferase is an enzyme that esterifies free cholesterol. A complete deficiency of this enzyme results in a diffusely cloudy cornea. This deficiency is thought to be transmitted as an autosomal recessive trait. We studied a family in which four members were homozygote recessive. In the homozygote recessive condition, a central corneal haze caused by deposition of numerous minute gray dots was consistently present. In the heterozygote condition, arcuslike changes were present in some of the patients studied. We found the corneal change in the recessive state to be sensitive and specific as a marker of this condition. Heterozygotes appear to have a higher incidence of arcuslike corneal changes.

Use of particle-loaded membranes to extract steroids for high-performance liquid chromatographic analyses improved analyte stability and detection.

Cortisol, cortisone, corticosterone, prednisone and prednisolone are extracted from serum using the novel particle-loaded octyl (C8)-bonded silica in PTFE membrane. Extracts are directly injected, without further concentration, onto a narrow (2.0 mm) or conventional (4.6 mm) bore octyldecyl (C18) HPLC column. Method performance data demonstrate linearity from 0.4 microgram/dl (low limit of detection) up to at least 60 micrograms/dl. Extraction recoveries exceeded 85% and precision (between-run) R.S.D.s averaged < 5%. Interferences were minimal and selectivity was improved over conventional immunochemical steroid assays. When compared to large particle sorbents packed in columns or to traditional liquid-liquid extractions, the membrane extracted steroids in less time, used less reagent, and had smaller elution volumes, thereby obviating steroid instability/adsorption problems associated with traditional concentrating techniques required to improve analytical sensitivity.

Concentrations of cyclosporin A and its metabolites in human tissues postmortem.

We report concentrations and distribution of cyclosporine A (CsA) and individual metabolites associated with various organ tissues and whole-blood specimens collected at autopsy from seven transplant patients who received CsA therapy. Solid-phase extraction (SPE) and specific high-performance liquid chromatographic (HPLC) procedures were used to separate and quantitate the cyclosporines. Patterns of deposition were unique for the various tissue types. Metabolites M17, M1, M18, and M8 (in addition to CsA) were the principal compounds detected in significant quantities. On a per weight basis, the sum concentration of CsA and metabolites in organ tissues was up to 53 times greater than in companion whole-blood specimens. Metabolite M17 prevailed in most tissues, except in fat and pancreas, where CsA was predominant. Overall, pancreas specimens contained a greater concentration of cyclosporines (per kilogram of tissue), followed consecutively by spleen, liver, fat, kidney, lung, bone marrow, heart, and whole blood. No CsA-related compounds were detected in brain or spinal cord tissue.

Application of a novel form of solid-phase sorbent (Empore membrane) to the isolation of tricyclic antidepressant drugs from blood.

We employed a new form of solid-phase material, the Empore octyl (C8) extraction membrane (SPEM), for the efficient extraction of tricyclic drugs from patients' serum specimens. Both extraction and companion high-performance liquid chromatographic (HPLC) assay of doxepin (DOX), desmethyldoxepin (DDOX), imipramine (IMI), desmethylimipramine (DESI), amitriptyline (AMI), nortriptyline (NOR), clomipramine (CLO), and desmethylchlomipramine (DCLO) are presented here. Routinely, serum (1.0 mL or less) adjusted to pH 5.5 with phosphate buffer is passed through the SPEM secured in a MF-1 microfilter unit. Proteins and potential interferences retained on SPEM are removed with an acetonitrile-water wash. The tricyclic drugs are eluted with HPLC mobile phase and the eluate is injected directly on a Zorbax cyanopropyl (CN) HPLC column, thereby avoiding time consuming evaporation-concentration steps that can affect drug stability. Recovery for all drugs exceeds 90% and analytical responses are linear from a lower limit of sensitivity of 8 micrograms/L up to at least 1000 micrograms/L. Between-run coefficients of variation (CV) range from 2.9 to 8.3% through the concentration range of 75 to 300 micrograms/L. Performance characteristics of the SPEM are compared to those of conventional large particle silica- and polymeric-based sorbents. Within the requirements of this assay, the SPEM extraction requires less sample volume and reduces elution and solvent volumes.

Correlations between scores on the Wechsler Intelligence Scale for Children-III and the General Purpose Abbreviated Battery of the Stanford-Binet IV.

Estimations of some relationships among scores on the "General Purpose Abbreviated Battery" of the Stanford-Binet: Fourth Edition and the Wechsler Intelligence Scale for Children-III were based on the responses of 14 boys and 18 girls enrolled in Grades 3, 4, and 5 and who took both tests. Of 13 Pearson correlations between the Binet IV composite score and the Wechsler subtest scores and IQs 12 were statistically significant (rs = .45 to .74). The new Wechsler scale appears to be a valid instrument for the 32 children (8-8 to 11-11) who were tested.

Cholesterol operational process specifications for assuring the quality required by CLIA proficiency testing.

Current U.S. governmental regulations and requirements for the quality of laboratory tests do not provide a consistent form, comparable numbers, or practical specifications for the routine operation of laboratory testing processes. For cholesterol, as an example, the Health Care Financing Administration provides an analytical performance criterion for proficiency testing to enforce the Clinical Laboratory Improvement Act (CLIA), whereas the U.S. National Cholesterol Education Program (NCEP) provides clinical guidelines for test interpretation, as well as analytical goals for imprecision and inaccuracy. Routine operating process specifications for imprecision, inaccuracy, and quality control can be derived from the analytical and clinical requirements for quality. Use of an analytical "total error" model and a clinical "decision interval" model provides logically consistent and numerically comparable specifications. Studies with these coherent models indicate that a cholesterol testing process properly planned to satisfy the CLIA analytical requirement will also satisfy the NCEP clinical requirement. To provide 90% assurance of detecting systematic shifts of a magnitude that would cause the CLIA analytical requirement to be exceeded, the operational specifications for a cholesterol testing process are an allowable CV of less than or equal to 2%, an allowable bias of less than or equal to 1%, and a control procedure with two measurements per run interpreted by 1(3)s, 1(2.5)s, or 1(3)s/2(2)s/R4s control rules.

Cholesterol--a model system to relate medical needs with analytical performance.

The evolution of cholesterol testing provides an example of a systematic approach that developed to relate the medical use of a laboratory test with the analytical performance requirements for that test. Laboratories today have the capability to perform cholesterol testing with the accuracy and precision necessary to meet medical needs. This statement can be made because (a) a standard diagnostic process has been established by the National Cholesterol Education Program; (b) an accuracy base is provided through a reference method that is readily available to manufacturers and laboratories; (c) the precision of analytical systems has been improved by manufacturers; (d) operating specifications for all such systems can be established, with statistical quality-control rules to ensure adequate within-run method performance; and (e) analytical performance is monitored by proficiency testing by using national quality requirements defined by CLIA '88 for acceptability. This cholesterol model provides a logical and scientific approach that should be applicable with other analytes to assure that the analytical performance of the laboratory test satisfies medical needs.

Influence of incomplete cholesteryl ester hydrolysis on enzymic measurements of cholesterol.

Esterolytic activity of three enzymic cholesterol reagents was evaluated with use of human serum-based reference materials. Primary cholesterol standards were used to calibrate the enzymic methods, and incomplete cholesteryl ester hydrolysis was measured as the bias between the enzymic cholesterol values and corresponding reference values. The average biases observed with eight reference specimens were 0.15, -1.16, and -7.24%, respectively, for a commercial enzymic reagent with microbial cholesterol esterase, a commercial reagent with an animal source of esterase, and a Centers for Disease Control (CDC) reagent preparation with a microbial cholesterol esterase. By liquid chromatography, we examined the respective specificities of the three esterase reagent preparations and found that the preparations differ in the rate and specificity of catalysis of cholesteryl ester hydrolysis. The CDC reagent was only partly active towards cholesteryl arachidonate, which probably accounts for most of the negative bias of this reagent. Esterolytic activity of the various enzymic reagents is related to the source and matrix of the cholesterol esterase and must be considered with respect to proper selection of calibration materials for serum cholesterol measurement.