Validation of a novel homogeneous assay for of HDL3-C measurement.

BACKGROUND: Low serum concentration of high density lipoprotein2 cholesterol (HDL2-C) is associated with increased risk of cardiovascular events. HDL2-C is calculated indirectly by subtracting high density lipoprotein3 cholesterol (HDL3-C) from total high density lipoprotein cholesterol (HDL-C). However, the special equipment and long assay times required for HDL3-C measurement have hindered the use of HDL2-C clinically. Here, we report the validation of a simple and rapid homogeneous assay for HDL3-C that is adaptable to clinical chemistry analyzers. MATERIALS AND METHODS: Method comparison based on 2740 serum specimens spanning the physiological range of HDL3-C was analyzed in singlicate to evaluate and validate a new homogeneous assay from Denka Seiken against the conventional dextran sulfate precipitation method. This study was performed over five days. Serum pools were prepared for the analysis of precision over 5 days (5 measurements per day), linearity, and interference (hemoglobin, bilirubin, and triglycerides) evaluation. RESULT: The homogeneous method had good within-run precision at concentrations of 24, 36, and 46 mg/dl, yielding standard deviations (SD) of 0.2 (0.9%) 0.4 (1.2%), and 0.5 (1.1%), respectively. Between-day precision, performed over 5 days using the same serum pools, yielded SD of 0.3 (1.4%), 1.0 (2.8%), and 0.9 (2.0%), respectively. The assay was linear from 1 to 100 mg/dl and correlated very well with the dextran sulfate precipitation method. There was no interference from hemoglobin up to 500 mg/dl, bilirubin up to 25 mg/dl, or triglycerides up to 1500 mg/dl. CONCLUSION: This homogeneous HDL3-C assay quantitatively measures HDL3-C in serum samples and has excellent precision, and can be implemented on an automated chemistry analyzer, thereby facilitating rapid measurement (~10 min) of a large number of samples in a standard clinical laboratory without the need for additional expensive equipment, laboratory space, or specially-trained staff.

Collaborative meta-analysis of individual participant data from observational studies of Lp-PLA2 and cardiovascular diseases.

BACKGROUND: A large number of observational epidemiological studies have reported generally positive associations between circulating mass and activity levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) and the risk of cardiovascular diseases. Few studies have been large enough to provide reliable estimates in different circumstances, such as in different subgroups (e.g., by age group, sex, or smoking status) or at different Lp-PLA2 levels. Moreover, most published studies have related disease risk only to baseline values of Lp-PLA2 markers (which can lead to substantial underestimation of any risk relationships because of within-person variability over time) and have used different approaches to adjustment for possible confounding factors. OBJECTIVES: By combination of data from individual participants from all relevant observational studies in a systematic 'meta-analysis', with correction for regression dilution (using available data on serial measurements of Lp-PLA2), the Lp-PLA2 Studies Collaboration will aim to characterize more precisely than has previously been possible the strength and shape of the age and sex-specific associations of plasma Lp-PLA2 with coronary heart disease (and, where data are sufficient, with other vascular diseases, such as ischaemic stroke). It will also help to determine to what extent such associations are independent of possible confounding factors and to explore potential sources of heterogeneity among studies, such as those related to assay methods and study design. It is anticipated that the present collaboration will serve as a framework to investigate related questions on Lp-PLA2 and cardiovascular outcomes. METHODS: A central database is being established containing data on circulating Lp-PLA2 values, sex and other potential confounding factors, age at baseline Lp-PLA2 measurement, age at event or at last follow-up, major vascular morbidity and cause-specific mortality. Information about any repeat measurements of Lp-PLA2 and potential confounding factors has been sought to allow adjustment for possible confounding and correction for regression dilution. The analyses will involve age-specific regression models. Synthesis of the available observational studies of Lp-PLA2 will yield information on a total of about 15 000 cardiovascular disease endpoints.

Atorvastatin inhibits calcification and enhances nitric oxide synthase production in the hypercholesterolaemic aortic valve.

OBJECTIVE: To study in a rabbit model the expression of endothelial nitric oxide synthase (eNOS) in association with the development of calcification of the aortic valve, and to assess the effects of atorvastatin on eNOS expression, nitrite concentration, and aortic valve calcification. METHODS: Rabbits (n = 48) were treated for three months: 16, forming a control group, were fed a normal diet; 16 were fed a 0.5% (wt/wt) high cholesterol diet; and 16 were fed a 0.5% (wt/wt) cholesterol diet plus atorvastatin (2.5 mg/kg/day). The aortic valves were examined with eNOS immunostains and western blotting. Cholesterol and high sensitivity C reactive protein (hsCRP) concentrations were determined by standard assays. Serum nitrite concentrations were measured with a nitric oxide analyser. eNOS was localised by electron microscopy and immunogold labelling. Calcification in the aortic valve was evaluated by micro-computed tomography (CT). RESULTS: Cholesterol, hsCRP, and aortic valve calcification were increased in the cholesterol fed compared with control animals. Atorvastatin inhibited calcification in the aortic valve as assessed by micro-CT. eNOS protein concentrations were unchanged in the control and cholesterol groups but increased in the atorvastatin treated group. Serum nitrite concentrations were decreased in the hypercholesterolaemic animals and increased in the group treated with atorvastatin. CONCLUSION: These data provide evidence that chronic experimental hypercholesterolaemia produces bone mineralisation in the aortic valve, which is inhibited by atorvastatin.

Cardiac troponin levels following monitored epileptic seizures.

Cardiac arrhythmia associated with myocardial injury is a proposed mechanism for sudden unexplained death in epilepsy. The authors measured serial cardiac troponin levels in 11 patients after monitored seizures. Using highly sensitive assays and criteria, no troponin elevations were seen, indicating that myocardial injury does not occur during uncomplicated seizures. An elevation in postictal troponin elevations should suggest the presence of cardiac injury secondary to neurocardiogenic mechanisms or primary cardiac factors, prompting further evaluation.

Urinary 11-dehydro-thromboxane B(2) and coagulation activation markers measured within 24 h of human acute ischemic stroke.

The aim of this study was to determine the extent of change in platelet and coagulation markers in the acute phase of ischemic stroke and to assess the utility of marker measurement in stroke subtype classification. Urinary 11-dehydro-thromboxane B(2) (11-dTXB2), a marker of in vivo platelet activation, and markers of coagulation activation, including prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and fibrinogen, were measured in 25 patients with ischemic stroke within 24 h of onset of symptoms. Marker levels in patients with ischemic stroke were compared with those in 19 age-matched controls who had not taken aspirin for at least 2 weeks before sampling and 25 healthy controls. Median marker levels were significantly increased in stroke over those in age-matched controls for fibrinogen (344 vs. 289 mg/dl; P=0.030), F1+2 (1.40 vs. 0.80 nmol/l; P=0.003), and TAT (6.65 vs. 2.20 microg/l; P<0.0001). Median marker levels for seven patients with cardioembolic stroke and 18 with non-cardioembolic stroke were not significantly different for any marker test. Eight patients taking aspirin at the time of the stroke had significantly lower 11-dTXB2 values than patients not taking aspirin (964 vs. 4,314 pg/mg of creatinine; P=0.007). Stroke patients not taking aspirin had significantly higher 11-dTXB2 concentration than age-matched controls (4,314 vs. 1,788 pg/mg of creatinine; P=0.006). Coagulation and platelet activation markers are increased in the acute phase of stroke regardless of the clinical mechanism. This finding suggests that the markers may not be useful for predicting clinical subtype of ischemic stroke in the acute phase.

Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry.

BACKGROUND: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. METHODS: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis. RESULTS: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. CONCLUSIONS: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.

Remnant lipoproteins induce endothelial plasminogen activator inhibitor-1.

Remnant lipoproteins (RLPs) accumulate in type III hyperlipoproteinemia, a condition associated with significant cardiovascular morbidity. The effect of RLPs on fibrinolysis is unknown. Our aim was to study the effect of RLPs on endothelial expression of plasminogen activator inhibitor-1 (PAI-1). After 24-h culture of human aortic endothelial cells with RLPs at concentrations of 0 (control), 0.038, or 0.076 mg triglyceride/mL, postculture PAI-1 antigen concentrations were: 870 +/- 80, 1963 +/- 183 (P = 0.005), and 3551 +/- 177 ng/mL (P < 0.001), respectively. Furthermore, after 24-h incubation of endothelial cells with RLPs (0 or 0.076 mg triglyceride/mL), PAI-1 activity increased from 0.667 +/- 0.144 to 1.268 +/- 0.198 U/mL, respectively (P = 0.008) and endothelial PAI-1 mRNA increased to 2.7 +/- 0.66 that of control (P = 0.048). In conclusion, RLPs from patients with type III hyperlipoproteinemia induce endothelial cell PAI-1 expression, which may contribute to a prothrombotic state.

Quantitative determination of plasma c8-c26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders.

We have developed a capillary gas chromatography-electron-capture negative-ion mass spectrometry (GC/MS) method for the quantitative determination of C8-C26 total fatty acids in plasma. Following hydrolysis, hexane extraction, and derivatization with pentafluorobenzyl bromide, fatty acid esters are analyzed in two steps: a splitless injection and a second, split injection (1:100) for the quantitation of the more abundant long-chain species. Fourteen saturated and 25 unsaturated fatty acids are quantified by selected ion monitoring in ratio to 13 stable-isotope-labeled internal standards. Calibrations exhibit consistent linearity and reproducibility. Intraassay (n = 17) and interassay (n = 12) CVs ranged from 2.5 to 13.2% and from 4.6 to 22.9%, respectively. Recoveries ranged from 76 to 106%. Reference ranges were established for four age groups (<1 month, 1 month to 1 year, 1-17 years, >18 years) and compared to specimens from patients with nutritional deficiency of omega-3 and omega-6 polyunsaturated fatty acids, inborn errors of mitochondrial fatty acid oxidation, and peroxisomal disorders. Retrospective evaluation of the concentration of linoleic acid in 35 cases with a diagnosis of essential fatty acid deficiency previously made by gas chromatographic analysis with flame ionization detection (GC/FID) found a specificity and sensitivity of only 55 and 50%, respectively, for the GC/FID method when compared to GC/MS.

Treatment of congenital erythropoietic porphyria in children by allogeneic stem cell transplantation: a case report and review of the literature.

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder of porphyrin metabolism in which the genetic defect is the deficiency of uroporphyrinogen III cosynthase (UIIIC). Deficiency of this enzyme results in an accumulation of high amounts of uroporphyrin I in all tissues leading to hemolytic anemia, splenomegaly, erythrodontia, bone fragility, exquisite photosensitivity and mutilating skin lesions. We describe the case of a 23-month-old boy who was cured of his CEP by a matched-sibling allogeneic bone marrow transplant, and review the published clinical experience regarding transplantation in this disease. He is alive and disease-free 15 months post transplant. All of his disease manifestations except for the erythrodontia have resolved. His UIIIC level and stool and erythrocyte porphyrin metabolites have almost completely corrected. He is the sixth child reported to be cured of this disease by stem cell transplantation, five cases being long-term survivors. If patients with this disease have an HLA-matched sibling, then stem cell transplantation should be strongly considered because this is currently the only known curative therapy.

Development of a rapid, quantitative method for LDL subfractionation with use of the Quantimetrix Lipoprint LDL System.

BACKGROUND: Recent evidence suggests that the presence of small, dense LDL is independently associated with increased risk of developing coronary artery disease. Current methods to subfractionate LDL are time-consuming and/or technically demanding. Therefore, we have sought the development of a less complex LDL subfractionation procedure. METHODS: LDL subfractions were separated using the Quantimetrix Lipoprint(TM) LDL System. High-resolution 3% polyacrylamide gel tubes were scanned densitometrically (610 nm) with a Helena EDC system. A computerized method to identify and quantitatively score the resolved LDL subfractions was developed. Results from the Quantimetrix method were compared using 51 plasma samples with values obtained by nondenaturing gradient gel electrophoresis (NDGGE) and nuclear magnetic resonance (NMR) spectroscopy. RESULTS: LDL subfractionation scores correlated significantly (P <0.05) with triglyceride, HDL-cholesterol, apolipoprotein B100, and LDL-cholesterol/apolipoprotein B100 (r = 0.591, -0.392, 0.454, and -0.411, respectively). For 51 samples, the Quantimetrix method classified 21 with small, 14 with intermediate, and 16 with large LDL. Of the 21 samples classified as small by Quantimetrix, 20 (95%) were classified as small (n = 18) or intermediate (n = 2) by NDGGE. All of the 16 specimens classified as large by Quantimetrix were either large (n = 14) or intermediate (n = 2) by NDGGE. LDL score was inversely correlated (r = -0.674; P <0.0001) with LDL particle size determined by NMR spectroscopy. CONCLUSIONS: A quantitative method for the assessment of LDL particle size phenotype was developed using the Quantimetrix Lipoprint LDL System. The method can be performed in less than 3 h in batch mode and is suitable for routine use in clinical laboratories.

Ascorbate levels in red blood cells and urine in patients with sickle cell anemia.

UNLABELLED: Ascorbic acid can be important in sickle cell anemia (SCA) because significant oxidative stress occurs in the disease. Ascorbate could contribute to reduction of the increased oxygen free radicals generated in sickle red blood cells (SRBC) and to the recycling of vitamin E in the cells, while renal loss could contribute to the low plasma levels. Evaluation of red blood cell (RBC) and urine ascorbate in SCA has not been reported. Results showed (1) ascorbate levels in SRBC were similar to those in normals; (2) urine ascorbate excretion was increased in 36% of patients; (3) plasma levels of ascorbate were decreased. CONCLUSIONS: (1) Ascorbate is present in SRBC, most likely due to ascorbate recycling, despite increased free-radical generation. (2) The increase in renal excretion may contribute to the low plasma levels of ascorbate. (3) The presence of ample ascorbate in SRBC and decreased plasma ascorbate suggests that ascorbate movement across the SRBC membrane may differ from normal RBC.

Silicon analysis of breast and capsular tissue from patients with saline or silicone gel breast implants: II. Correlation with connective-tissue disease.

The silicone breast implant controversy rages on. Recent work has demonstrated that normal or baseline breast tissue silicon levels in women who had had no prior exposure to any type of breast implant may be as high as 446 microg/gm of tissue. These data ranged from 4 to 446 microg/gm of tissue, with a median of 27.0 microg/gm of tissue. In addition, numerous other epidemiologic and rheumatologic studies have demonstrated no association between silicone breast implants and any connective-tissue diseases. Despite these reports, the use of silicone implants remains restricted. The present study measured breast and capsular tissue silicon levels from 23 breasts in 14 patients with saline implants, and from 42 breasts in 29 patients with silicone implants. No patient in the saline implant group presented with signs or symptoms of connective-tissue disease. Patients with silicone implants, however, were divided into three groups based on the presence or absence of signs or symptoms of connective-tissue disease: group I, no symptoms or signs; group II, + symptoms, no signs; and group III, + symptoms, + signs. Six patients in group III were diagnosed with a specific connective-tissue disease, including systemic lupus erythematosus, rheumatoid arthritis, or scleroderma. The most common indications for implant removal or exchange were capsular contracture and implant rupture, although 41 percent of patients with silicone implants expressed media-related concern over the implant issue. The most common symptoms described by patients in groups II and III were joint pain and stiffness, arm pain and numbness, and fatigue. In all groups, capsular tissue silicon levels were significantly greater than breast tissue levels. This finding may indicate that the capsule serves as a barrier to the distribution of silicone from the implant into adjacent breast tissue. Although breast tissue silicon levels in patients with silicone implants were not significantly greater than those in patients with saline implants (p = 0.48), capsular tissue levels in patients with silicone implants were, indeed, significantly greater than those in patients with saline implants (p < 0.001). However, no statistically significant differences in tissue silicon levels were observed with relation to the presence or absence of connective-tissue disease signs or symptoms in patients with silicone implants (groups I to III). Therefore, these data strengthen the conclusion that there is no association between tissue silicon levels and connective-tissue disease.

Determination of silicon in breast and capsular tissue from patients with breast implants performed by inductively coupled plasma emission spectroscopy. Comparison with tissue histology.

A method for analysis of silicon in tissue was developed to determine silicon content in breast parenchymal and periprosthetic capsular tissues of patients with silicone or saline implants and to compare levels in tissues from normal (nonaugmented) breasts. It is of interest to determine whether increased silicon content in tissues can be associated with morbidity in patients who have received silicone implants. This manuscript addresses the issues involved in analysis of breast tissue samples for silicon and compares silicon levels with tissue histologic findings and patient morbidity. One hundred sixty tissue samples were obtained for silicon analysis from 72 patients during augmentation, capsulectomy with or without replacement mammoplasty, mastectomy, or biopsy procedures and were frozen in acid-washed polystyrene tubes at 220 degrees C until analysis. Samples were thawed, sectioned to approximately 0.1 g (dry weight), and digested in nitric acid before analysis by inductively coupled plasma emission spectroscopy, monitoring emission intensity at 251.6 nm. Tissue silicon levels (breast parenchymal and periprosthetic capsular tissue) in patients with silicone gel implants were much higher (mean, 9,287 micrograms/g, n = 106) than in patients with saline implants (mean, 196 micrograms/g, n = 37) or nonaugmented breasts (mean, 64 micrograms/g, n = 17). Histologic examination was performed on 54 tissue samples stained with hematoxylin-eosin. Tissue samples were rated as to degree of inflammation and calcification, and amount of giant cells, foamy histiocytes, and vacuoles containing a colorless refractory material. Vacuolization and foamy histiocyte ratings correlated significantly with tissue silicon concentration. No correlations were found between tissue silicon concentration and inflammation, calcification, or giant cell rating. Implant age (number of years an implant was in place before sampling) correlated with capsular tissue silicon concentration in patients with intact implants but not in those with ruptured implants. No difference in tissue silicon concentration was found between patients with or without signs or symptoms of morbidity. Using 0.1 g of tissue, the method was linear to 1,000 micrograms/g, and sensitivity was 3.7 micrograms/g. Precision between runs (mean, 5.1 micrograms/g; coefficient of variance, 13.7%; n = 13) was calculated from multiple analyses of a bovine liver standard (National Bureau of Standards, reference material 1577a). Significant biologic variability (21.4% to 52.5%) was seen in tissues with high silicon levels. Paraffin-embedded, formalin-fixed tissues are not amenable to silicon analysis by this method, because of leaching of silicone from the tissues during preparation. Thus only fresh frozen tissue samples were used.

Silicon analysis of breast and periprosthetic capsular tissue from patients with saline or silicone gel breast implants.

The ubiquitousness of silicon is well known. Recent work has demonstrated measurable baseline levels of silicon in nonaugmented cadavers, subsequent to numerous reports of significant elevations of such levels within patients with silicone breast implants and even more reports alleging a causal relation between silicone gel prostheses and connective-tissue diseases. Despite the lack of scientifically substantiated data that such a relation exists, the calamitous silicone breast implant controversy has ensued. Saline-filled breast implants are constructed with a silicone elastomer envelope that remains in direct contact with periprosthetic capsular tissue following implantation. Although there is no evidence to link saline implants with any disorders, it is important to know if saline breast implants contribute any silicon to human body baseline silicon levels. The present study measured tissue silicon levels in 28 breasts of 16 patients with saline-filled implants to determine if the silicone envelope of these prostheses can contribute to the elevation of such levels. These data were compared with data from 116 breasts of 65 patients with silicone gel-filled prostheses as well as breast tissue from 17 patients (controls) who had never been exposed to either type of implant. Samples of breast tissue and periprosthetic capsular tissue were obtained from patients with both intact and ruptured implants. Silicon levels of breast tissue specimens from patients with saline-filled implants were within the range of the controls if the implants were intact. Silicon levels in periprosthetic capsular tissue from patients with intact saline-filled implants were significantly higher than controls (p < 0.02); however, they were still 100-fold less than capsular tissue levels from patients with intact gel-filled implants. Silicon levels measured in both types of tissue were significantly elevated in patients with silicone gel-filled implants compared with controls (p < 0.01). In the case of ruptured gel implants, breast tissue demonstrated higher silicon levels than did similar specimens from patients with intact implants (p < 0.054); periprosthetic capsular tissue levels also were elevated, although the differences were not statistically significant (p = 0.54). These findings are independent of the implant brand or length of exposure to the particular prosthesis. The finding of elevated levels of silicon in both breast and periprosthetic capsular tissue in patients with silicone gel-filled implants in no way implies or substantiates any claim of a causal relationship between silicone and any reported illnesses.

Arthroscopic transglenoid suture capsulolabral repairs: preliminary results.

The arthroscopic transglenoid suture technique was performed on 38 shoulders for anterior capsulolabral repair or reconstruction. The primary complaint was instability in 34 shoulders (89%) and pain in 4 shoulders (11%). In the instability subgroup, 3 (9%) experienced instability in their sleep, 17 (50%) with activities of daily living, and 14 (41%) with athletic activities. Arthroscopic examination revealed labral detachment in 35 shoulders (92%) with additional capsular abnormalities noted in 17 (45%). The remaining 3 shoulders (8%) demonstrated capsular laxity and thinning without labral detachment. Twenty-nine shoulders had complete relief of instability. There were no redislocations. Four shoulders (10%) had recurrence of instability. Twenty shoulders (53%) obtained full range of motion, 15 (39%) had minor (< 10 degrees) loss of external rotation, 2 (5%) experienced greater (> 10 degrees) loss of external rotation, and 1 improved over a restricted preoperative range of motion. Fifteen of the 20 competitive athletes and 11 of the 15 recreational athletes returned to the same level and same type of athletic activity. Five patients did not resume their preinjury athletics because of unrelated life-style changes; they reported no shoulder problems. Four patients significantly reduced their athletic participation because of postoperative instability or residual pain. In conclusion, relief of apprehension, reestablishment of shoulder stability and return to athletic activity, including contact and throwing activities, can be achieved safely and effectively in appropriately selected patients.

Determination of fibrinogen in plasma by high-performance immunoaffinity chromatography.

A high-performance immunoaffinity chromatographic (HPIAC) method for fibrinogen was developed which had several advantages over existing methodologies including increased linear range and no interference from heparin. Several modifications of usual HPIAC procedures were necessary including the employment of a methacrylate polymeric support to reduce non-specific adsorption and the addition of urea to a pH 2.1 elution buffer to affect elution. A significant split-peak effect (i.e., unretained fibrinogen) was noted at higher flow-rates and at higher fibrinogen concentrations, which was shown to be temperature-dependent, with the amount of fibrinogen retained on the column increasing with increased temperature.