Monthly intra-individual variation in lipids over a 1-year period in 22 normal subjects.

OBJECTIVES: Previous reports on biological variation in lipids differ widely in the time interval between sampling, the number of samples analyzed per patient and the total study period. The present investigation was carried out to determine monthly intra-individual variation in lipids over 1 year and to establish whether there was a consistent change in lipid values over the summer months. The importance of taking this variation into consideration during the assessment of risk of coronary heart disease (CHD) was also examined. DESIGN AND METHODS: Cholesterol, triglycerides, HDL, apo A1, and apo B were measured at monthly intervals for 12 months in 22 healthy, free-living volunteers (11 females, 11 males) by standardized methods. RESULTS: When compared to analytical variation, biological variation was the dominant component of the intra-individual changes observed during the 1-year study period. As expected, triglycerides showed the greatest biological variation; the ratio of biological/analytical variation was 33.1. Much smaller ratios were observed for the other lipids measured in this study with values ranging from 4.2 to 6.8. Different subjects attained their maximum and minimum values in virtually every month of the year. There were significant reductions in cholesterol, HDL, LDL, and apo A1 in the summer months while triglycerides showed a non-significant increase and apo B a non-significant decrease during this period. CONCLUSIONS: All the analytes showed considerable intra-individual variation. It is, therefore, important to measure lipids sequentially over several weeks to arrive at an average value for risk stratification for CHD.

An overview of quality control practices in Ontario with particular reference to cholesterol analysis.

BACKGROUND: The Laboratory Proficiency Testing Program (LPTP) assesses the analytical performance of all licensed laboratories in Ontario. The LPTP Enzymes, Cardiac Markers, and Lipids Committee conducted a "Patterns of Practice" survey to assess the in-house quality control (QC) practices of laboratories in Ontario using cholesterol as the QC paradigm. DESIGN AND METHODS: The survey was questionnaire-based seeking information on statistical calculations, software rules, review process and data retention, and so on. Copies of the in-house cholesterol QC graphs were requested. A total of 120 of 210 laboratories were randomly chosen to receive the questionnaires during 1995 and 1996; 115 laboratories responded, although some did not answer all questions. RESULTS: The majority calculate means and standard deviations (SD) every month, using anywhere from 4 to >100 data points. 65% use a fixed mean and SD, while 17% use means calculated from the previous month. A few use a floating or cumulative mean. Some laboratories that do not use fixed means use a fixed SD. About 90% use some form of statistical quality control rules. The most common rules used to detect random error are 1(3s)/R4s while 2(2s)/4(1s)/10x are used for systematic errors. About 20% did not assay any QC at levels >5.5 mmol/L. CONCLUSIONS: Quality control data are reviewed daily (technologists), weekly and monthly (supervisors/directors). Most laboratories retain their QC records for up to 3 years on paper and magnetic media. On some QC graphs the mean and SD, QC product lot number, or reference to action logs are not apparent. Quality control practices in Ontario are, therefore, disappointing. Improvement is required in the use of clinically appropriate concentrations of QC material and documentation on QC graphs.

Blood lipid profiles in children with acute lymphoblastic leukemia.

BACKGROUND: Abnormal blood lipid profiles have been associated with cancer. The objective of this study was to investigate the frequency and clinical significance of altered lipid profiles in children with acute lymphoblastic leukemia (ALL), the most common form of malignant disease in this age group. METHODS: Fasting blood lipid profiles (cholesterol [C], triglycerides [TG], high density lipoprotein [HDL], low density lipoprotein, very low density lipoprotein, apolipoproteins A1 [apo A1] and B, and lipoprotein a [Lp(a)]) were obtained in 24 children with ALL at diagnosis, 16 children during consolidation therapy with L-asparaginase, and 18 children during maintenance therapy without L-asparaginase. For comparison the authors studied lipid profiles in 15 children previously treated for leukemia, 15 healthy control children, and 17 children with other forms of cancer, both localized and widespread. RESULTS: An altered blood lipid profile was observed at the time of diagnosis of ALL. Statistically significant values included elevated TG (1.82+/-1.23 mmol/L), reduced HDL-C (0.54+/-0.24 mmol/L), and reduced ApoA1 (0.77+/-0.18 g/L) levels. A wide range of Lp(a) levels (0-1990 mg/L) were observed. Significantly reduced HDL-C (0.55+/-0.20 mmol/L) and ApoA1 (0.69+/-0.22 g/L) were observed in children with widespread but not localized solid tumors at diagnosis. C and TG correlated with serum albumin levels. Significant therapy-related changes in lipid profiles were observed in children with ALL during combination therapy with L-asparaginase (extremely elevated TG levels [3.34+/-2.82 mmol/L] and a striking reduction in Lp(a) levels) that were not observed during combination therapy without L-asparaginase or in children during treatment for solid tumors. In this small study there was no relation between these abnormalities and either thromboembolic events or pancreatitis. Blood lipid profiles in children with ALL returned to normal on completion of therapy. CONCLUSIONS: The lipid abnormalities observed at diagnosis in children with widespread cancer (ALL or solid tumors) may reflect altered nutritional states or altered lipid metabolism. Reduced concentrations of Lp(a) and elevated TG levels suggest L-asparaginase specific alterations and may provide insight into the toxicity associated with this drug.

Proficiency testing of creatine kinase and creatine kinase-2: the experience of the Ontario Laboratory Proficiency Testing Program.

The Ontario Laboratory Proficiency Testing Program has regularly monitored the analytical performance of total creatine kinase (CK) (approximately 230 participants) and CK isoenzyme-2 (CK-MB) (approximately 160 participants) throughout the entire province. Consistently, a wide dispersion of results has been observed not only between different analyzer systems but also among identical analyzers. Accordingly, the results of the last three proficiency surveys for these analytes were examined statistically to establish both the extent of these variations and the range of values reported for the male upper reference ranges. The results of many of the analyzer systems were significantly different from each other, as were many of the reference ranges. This unsatisfactory situation may only be remedied by the use of reference materials as shown by others. The consequences of these findings also effect the reliability of epidemiological surveys such as the WHO MONICA Project (Circulation 1994;90:583-612), which monitors deaths due to heart disease and includes cardiac enzyme results in its criteria.

Mutations in cholesteryl ester transfer protein and hepatic lipase in a North American population.

OBJECTIVE: To examine a North American population sample with increased HDL cholesterol for mutations in the genes coding for cholesteryl ester transfer protein (CETP) and hepatic lipase (HL). DESIGN AND METHODS: Seventy individuals with increased HDL cholesterol at the time of initial presentation to the Lipid Clinic (males > 1.7 mmol/L, females > 1.8 mmol/L) were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) analysis for known mutations in CETP intron 14 and exon 15 and HL exons 6 and 8. RESULTS: CETP intron 14 mutation frequency 0.7%, CETP exon 15 A1503G 0%, HL exon 6 C873T 2.1%, HL exon 8 C1221T 0%. An unusual mutation in CETP exon 15 G1533A was found at a frequency of 3.5%. The sequence of this mutation was determined to be a G to A change at bp 1533 resulting in a predicted amino acid change of arginine to glutamine at position 451. CONCLUSIONS: Known mutations in CETP were much less prevalent in this North American population than in the Japanese populations that have been previously reported. HL mutations, described previously in only 6 families worldwide, appear to be more prevalent than previously recognized. CETP G1533A, reported only once previously is prevalent in this population at a surprisingly high frequency. The functional significance of this mutation is unknown.

Monthly intra-individual variation in lipoprotein(a) in 22 normal subjects over 12 months.

OBJECTIVES: It is generally believe that lipoprotein(a) (Lp(a)) levels remain relatively constant in the same individual, but there is a paucity of data to substantiate this belief. This study was undertaken to determine the extent of intra-individual variation in Lp(a) over a 12-month period. DESIGN AND METHODS: Lp(a) was measured monthly in duplicate over a 12-month period in 11 females and 11 males who were healthy, free-living, normal subjects by the Incstar Immunoprecipitin method using a goat antibody which was monospecific for Lp(a). RESULTS: Some subjects showed considerable month-to-month variations which were not correlated with changes in other lipid parameters or with weight. Others showed fairly constant Lp(a) levels, with a few values which were quite different from the rest. This was not attributable to methodological factors; low and high controls gave mean (mg/L), SD and CV values of 181, 8.6, 4.7 and 431, 14, 3.3, respectively. The difference between the minimum and maximum values in the same individuals ranged from a low of 14 mg/L in one subject to a high of 229 mg/L in another over the one-year period. CONCLUSIONS: Lp(a) showed greater intra-individual variations in normal subjects than is commonly believed. It is therefore recommended that Lp(a) should be measured sequentially over a few weeks to arrive at a mean value for assessing risk of coronary heart disease.

A double-blind randomised controlled trial of the effects of medroxyprogesterone acetate on bone density of women taking oestrogen replacement therapy.

OBJECTIVE: To assess the effects of medroxyprogesterone acetate on bone density in women who have had a hysterectomy. DESIGN: Randomised, double-blind, placebo-controlled trial of medroxyprogesterone acetate 10 mg, 20 mg or placebo as an adjunct to oestrogen therapy. PARTICIPANTS: One hundred and twenty-three women, aged 18 to 45 years and currently receiving daily oestrogen, who presented at a university-based rheumatology practice. INTERVENTIONS: The women were randomly assigned to receive either medroxyprogesterone acetate 10 mg, 20 mg or placebo daily beginning on day 15 of each month for one year. Forty-one women were randomised into each group. MAIN OUTCOME MEASURE: The primary outcome measurement was the percentage of change from baseline in bone mineral density of the lumbar spine (L2-L4). Secondary outcome measures included differences in femoral neck bone density, cholesterol and triglyceride levels between groups. RESULTS: At one year, change in bone mineral density did not differ between either the treatment or placebo groups. Medroxyprogesterone acetate 20 mg and 10 mg led to statistically significant reductions in very low density lipoprotein cholesterol, total triglycerides, and very low density lipoprotein triglycerides when compared with placebo. Medroxyprogesterone acetate 20 mg also led to a statistically significant reduction in high density lipoprotein cholesterol, high density lipoprotein-2 cholesterol, and high density lipoprotein-2 triglycerides. CONCLUSIONS: Medroxyprogesterone acetate at either dose as an adjunct to oestrogen did not improve bone mineral density at one year when compared with placebo. Medroxyprogesterone acetate 10 mg may not adversely affect lipids. Medroxyprogesterone acetate 20 mg, however, did reduce high density lipoprotein cholesterol and therefore may increase cardiovascular risk.

Evaluation of a turbidimetric procedure for apolipoproteins A1 and B on the Ciba Corning 550 EXPRESS.

We evaluated a turbidimetric method for the estimation of apo A1 and apo B on the Ciba Corning 550 EXPRESS using Ciba Corning reagents. Interference due to bilirubin, hemoglobin, lipemia, triglycerides, and uremia was minimal, with apolipoproteins (apo) A1 and B results usually within +/- 4% of expected values. Within-run and day-to-day imprecision (coefficients of variation) ranged from 1.96 to 3.60% and 2.63 to 3.39% for apo A1 and 1.02 to 1.74% and 2.08 to 3.66% for apo B, respectively. Accuracy was determined by participation in the IFCC apolipoprotein standardization project in which results obtained on 50 patient samples were compared to those obtained by the reference laboratory. Apo A1 and apo B showed an average bias of +3.7% and +2.0% and correlation coefficients of 0.986 and 0.977, respectively. Results were also compared to those obtained on the Behring Turbitime system and showed a bias of +7.5% and -8.8% for apo A1 and apo B, respectively. The Ciba Corning automated method was rapid and gave good accuracy, precision, linearity, and parallelism and was relatively unaffected by raised triglyceride values.

Variation in human blood high-density lipoprotein response to oral vitamin E megadosage.

In a preliminary attempt to distinguish random intrinsic and methodologic variations of blood lipid levels from any that have possibly been induced by ingestion of vitamin E, blood samples were analyzed in duplicate for lipids several times before and during oral vitamin E administration. Three of eight subjects showed temporally closely coordinated and maintained increases of high-density lipoprotein cholesterol and apolipoprotein-A, total apolipoprotein-A, and the ratio of high-density to low-density cholesterols. Changes of other lipids in these three subjects, and lipid changes in the other subjects, were much less dramatic, fell within the range of intrinsic random variation or statistical uncertainty, and showed no significant trends. The results suggest that elevation of high-density lipoproteins in response to ingestion of megadoses of vitamin E is very much an individual characteristic and not uniformly typical of the population at large. The findings complement published examples of individual variation of response to vitamin E as an explanation of disagreements between other reports.

Role of lipoxygenase metabolism in platelet function: effect of aspirin and salicylate.

Aspirin inhibits thromboxane A2 (TxA2) production whereas its salicylate moiety inhibits 12-hydroxy-eicosatetraenoic acid (12-HETE) production in the platelet. The significance of the latter effect on platelet function is unclear. We examined the effects of aspirin and salicylate on (i) platelet/collagen adhesion using 3H-adenine-labelled human platelets and collagen-coated discs, (ii) platelet aggregation induced by thrombin, collagen, ADP and arachidonic acid, and (iii) platelet TxA2 and 12-HETE synthesis as measured by radioimmunoassay and high pressure liquid chromatography respectively. Aspirin (50 uM) decreased platelet aggregation and increased platelet adhesion. The decrease in aggregation was associated with inhibition of TxA2 production and the increase in adhesion was associated with enhanced 12-HETE production. Salicylate had the opposite effects. Platelet aggregation was increased and platelet adhesion decreased. The increased aggregation was associated with enhanced TxA2 production and the decrease in aggregation was associated with inhibition of 12-HETE production. These observations suggest that 12-HETE facilitates platelet adhesion which can be altered by salicylate treatment.

Endothelial cells produce a lipoxygenase derived chemo-repellent which influences platelet/endothelial cell interactions--effect of aspirin and salicylate.

We performed experiments to determine whether endothelial cells synthesize phospholipid metabolites via the lipoxygenase pathway and whether these metabolites influence platelet/vessel wall interactions. Monolayers of cultured human endothelial cells were incubated with 14C-arachidonic acid and their cyclo-oxygenase and lipoxygenase metabolites were extracted and identified by radioimmunoassay, thin layer chromatography and high performance liquid chromatography. We found that in addition to the membrane-associated production of PGI2, endothelial cells synthesized a cytosol-associated metabolite, LOX, which was presumably derived through the lipoxygenase pathway. Inhibition of LOX was associated with an increase in PGI2 production and inhibition of PGI2 with an increase in LOX production. Under either condition, platelet adhesion to cultured endothelial cells was significantly decreased. In contrast, when both PGI2 and LOX production were inhibited, platelet adhesion to endothelial cells was enhanced. Furthermore, when LOX was bound to a thrombogenic surface, platelet adhesion was significantly decreased whereas when arachidonic acid or 12-HETE was bound to the surface, platelet adhesion was increased. We conclude that endothelial cells produce not only a cyclo-oxygenase metabolite, but also a lipoxygenase metabolite, both of which influence platelet/endothelial cell interactions.

Lipoidal bladder concretion: an unusual autopsy finding in acute lymphoblastic leukemia.

The occurrence of a large (2.3 gm.), wax-like, creamy white concretion in the bladder of a 7-year-old girl who died of acute lymphoblastic leukemia is reported. Lipids constituted 40% of the total weight and triglycerides accounted for 95% of the total lipids. The fatty acid composition of the individual lipid classes showed a predominance of saturated fatty acids (75 to 80%). Factors influencing the pathogenesis of the material are discussed. A review of the literature on childhood leukemia showed no reference to such lipid abnormalities and the occurrence of this type of concretion has not been reported previously.

Evaluation of an automated immunoassay procedure for apo-LDL for phenotyping type II hyperlipoproteinemia.

Apo-low density lipoproteins were determined by an automated immunoassay procedure on serum samples from 88 normolipidemic individuals and 84 hyperlipoproteinemic subjects, to establish whether this method was useful in the routine detection of type II hyperlipoproteinemia. The results obtained were compared with the cholesterol levels of the same specimens. In subjects with type II hyperlipoproteinemia, the apo low density lipoprotein levels, as well as the ratio of low density lipoprotein cholesterol/apo-low density lipoprotein were higher, as expected, than in normals or in subjects with other types of hyperlipoproteinemia. However, there was considerable overlap in individual values of both these parameters, between patients with type II hyperlipoproteinemia and normals or subjects with other types of hyperlipoproteinemia, suggesting that apo low density lipoprotein levels alone were not sufficiently discriminatory for the laboratory determination of type II hyperlipoproteinemia.

Xanthoma disseminatum. Clinical, metabolic, pathologic, and radiologic aspects.

A 55-year-old man with the classical mucocutaneous lesions of xanthoma disseminatum has been followed up for a period of 13 years. The special features of this case, which make it unique, are as follows: (1) the availability of histologic data on multiple lesions for more than a ten-year period; (2) the progressive nature of the multiple osseous lesions; (3) the metabolic studies that show no evidence for accumulation of abnormal sterols in a xanthoma, the blood, or intestinal aspirate; (4) the development of hypothyroidism and symptoms or signs, or both, of an intracerebral and an intraspinal lesion; (5) the partial regression of the cutanous symptoms and lesions while receiving clofibrate, in spite of progression of the mucous membrane and osseous lesions, and (6) the failure to develop diabetes insipidus to date.

Fatty acid composition of margarines.

The fatty acid composition of 27 samples of bar margarines, 58 samples of tub margarines, and one sample of a liquid margarine spread, purchased at different centers in Canada and the United States over a 1-year period (1973-1974), has been determined. The values of total polyunsaturated fatty acids determined by gas-liquid chromatography were compared to the results obtained by an enzymatic method using lipoxidase. The margarines have also been compared on the basis of fatty acid composition and polyunsaturated to saturated fatty acid ratios. All of these parameters showed considerable variations among the different samples analyzed in this study. As a general rule, soft (tub) margarines tended to have a higher concentration of cis,cis-9,12-octadecadienoic (linoleic) acid than hard (bar) margarines. The labeling of the products as regards fatty acid composition was not always helpful in choosing a margarine of high linoleic acid composition.

Effect of dietary di-2-ethylhexyl phthalate on lipid biosynthesis in selected tissues from the rat, in vitro.

Di-2-ethylhexyl phthalate (DEHP), a plasticizer commonly used in the production of polyvinyl chloride plastics, has become an environmental pollutant. At the present time, the biological significance of phthalates in the environment is unknown. In the present studies, we observed that addition of DEHP to a stock diet of rats resulted in marked effects on incorporation of 14C-acetate into lipid by liver and kidney slices; other organs, such as heart, testes, and aorta, were unaffected. Incorporation of 14C-acetate into total lipid of liver (dpm/mg wet wt) from rats fed 0.5% or 1.0% DEHP for 10 or 18 days, respectively, was decreased to ca. 50% of control values. The decreased incorporation into liver lipid is not attributable to any one lipid fraction, inasmuch as incorporation into the phospholipid, sterol + diglyceride, free fatty acid, triglyceride, and sterol ester + hydrocarbon fractions was decreased 30-70% with respect to controls. In addition, the percent distribution of 14C-acetate among the individual phospholipids was ca. 25% lower in phosphatidyl choline of the DEHP-fed rats. In rats fed 0.5% DEHP, incorporation of 14C-acetate into total lipid of kidney was similar to control values, but incorporation into the triglyceride and sterol ester + hydrocarbon fraction was decreased 30-40%, whereas incorporation into the sterol + diglyceride fraction was increased 38%. Livers from DEHP-fed rats were ca. 20% larger than livers from control rats and, at the 0.5% level of DEHP feeding, testes wts were elevated; no significant changes were noted in wts of spleen, heart, aorta, kidney, or body wt gains in rats fed DEHP. These studies emphasize a subtle toxicity of phthalate esters not previously reported and emphasize the need for further biochemical studies to evaluate the effect of phthalates on biological systems.