Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Erythrocyte CuZn Superoxide Dlsmutase and the Extent of Coronary Atherosclerosis from coronary angiography (5). Erythrocyte CuZn SOD activity was determined by a previously described method (6). The statistics were performed by t-test and correlation regression analysis. Compared with the controls, the patients tended to have higher total cholesterol and triglyceride concentrations and lower high-density lipoprotein and lower ejection fractions (Table 1). The patients also tended to have lower CuZn SOD values, but the difference between the two groups was not significant statistically (P >0.05). The correlation between CuZn SOD activity and CAD was negative and weak (r = 0.2), as was the correlation between CuZn SOD and the number of vessels affected (r = 0.16). The correlation between the concentrations of CuZn SOD and cholesterol was weak but significant (r = 0.3, P <0.05). There was no correlation between CuZn SOD and other lipid variables. The negative findings in our study, the lack of a significant trend towards lower CuZn SOD values in patients with atherosclerosis, and no correlation between CuZn SOD activity and the extent of atherosclerosis as judged by the coronary score or ejection fraction may be due to the small number of patients studied. Other factors affecting erythrocyte SOD may be operative, despite our attempt to exclude clinical situations that affect SOD activities. Also, erythrocyte CuZn SOD may not adequately indicate the tissue CuZn SOD activity in the body. Other indices of free radical activity, e.g., glutathione, catalase, plasma thiols, or plasma malondialdehyde, which were not measured in this To the Editor: Superoxide dismutases (SODs) are considered important for protecting living cells against toxic oxygen derivatives, e.g., lipid peroxides, which have been implicated in the initiation of atherogenesis and coronary artery disease (CAD) (1). Recent trials, showing that several antioxidants delay or prevent the progression of atherosclerosis, support this implication (2). Moreover, preliminary experiments have shown that SOD, catalase, and glutathione peroxidase might be biological indicators of chronic diseases such as diabetes, alcoholism, and cancer (3). Erythrocytes have some of the highest CuZn SOD content of any tissue in the human body (4). Possibly, therefore, CuZn SOD activity in a patient may be an important marker for the development of atherosclerosis. To determine whether erythrocyte CuZn SOD activities are a biological marker of atherosclerosis, we determined these values in patients with angiographically proven CAD and compared them with those in healthy controls. Patients with other disease states that alter SOD activity, such as diabetes, renal disease, anemia, altered thyroid states, obesity, alcoholism, and smoking, were excluded. We studied 43 patients with angiegraphically proven CAD (36 men, 7 women, mean ages 51± 10 years) and 31 healthy controls matched for Body Mass Index with the patients (12 men, 19 women, mean ages 47 ± ii years). The coronary scores were determined study, may be more important markers. We conclude that we could not demonstrate that erythrocyte CuZn SOD was an important marker of coronary atherosclerosis. Further studies in a larger group of patients are needed to clariir this matter. References 1. Stringer MD, Gorog PG, Freeman A, Kjikksor VV. Lipid peroxides and atherosclerosis. Br Med J 1989;298:281-4. 2. Stampfer MJ, Hennekens CH, Manson JE, Colditz AG, Rosner B, Wfflett C. Vitamin E consumption and the risk of coronary disease in women. N Engi J Med 1993;328:1444-8. 3. Guemouri L, Artur Y, Herbeth B, Jeandel C, Cuny G, Siest G. Biological variability of superoxide dismutase, glutathione peroxidase, and catalase in blood. Clin Chem 1991;37:1932-7. 4. Marklund S. Distribution of CuZn superoxide dismutase and Mn superoxide diamutase in human tissues and extracellular fluids. Acta Physiol Scand 1980;492:19-23. 5. Gensini GO. Coronary arteriography. Mount Kis, CO: Futura Publishing, 1973: 269-74. 6. Winterbourn CC, Hawkins ER, Brian M, Carrell WR. The estimation of red cell superoxide dismutase activity. J Lab Clin Med 1972;85:337-41. Fatih Sinken S Lale Tokgozoiu’ Nurten Renda Sel#{231}uk Adabag Dept. of Cardiol. and Biochem. Hacettepe University Faculty of Med. Hacettepe, Ankara, Turkey ‘Author for correspondence. Erroneous Results Emit#{174} Reagents Table 1. Laboratory and anglographic data of the patients Mean ± and controls. SD Patients Controls 43 31 CuZn SOD, U/g Hb Cholesterol, mg/L Triglyceride, mg/L 3475 ± 1018 2070 ± 630 2050 ± 1370a HDL, mg/L LDL mg/L 1240 ± 510 3618 ± 1042 1900 ±450 1120 ± 620 570 ± 150 1110 ± 550 62± Ejection fraction, % Coronary score Body Mass Index 450 ± 56 7 ± 26 ± 120 17a ± 4a 3 ‘Significantly different from controls: P <0.05. Hb, hemoglobin; HDL high-densitylipoprotein; LDL, low-density lipoprotein. with Diluted 0 24±6 To the Editor: Previous reports high concentrations tate tem sults with CA). dehydrogenase samples with (1, 2) have linked of lactate and lac(LD) in postmor- false-positive refor ethanol screening performed Emit#{174} assays (Syva, Palo Alto, Enzyme and substrate were apparently present in sufficient quantities to convert NAD to NADH and generate a signal. Here we report falsely low Emit-measured drug concentrations associated with high LD activity in serum. A serum specimen from an adult in- _________ CUNICAL CHEMISTRY, Vol. 40, No. 8, 1994 1597 tensive-care patient was analyzed for total phenytoin with Emit homogeneous enzyme assay reagents diluted 20-fold as previously described (3, 4). Briefly, reagent A, supplemented with NAD and glucose 6-phosphate to 4.5 mmol/L each, was added at time 0, and after 5 mm reagent B was added; the first absorbance reading was taken at 6 mm and the final reading at 10 miii. A serum ultrafiltrate was prepared by centrifugation through a Centrifree#{174} device (30-kDa-cutoff YMT membrane; Amicon Div., W. R. Grace, Beverly, MA) (4), and free phenytoin was measured with undiluted Emit reagents, according to the manufacturer’s instructions. The total phenytoin measured was 5.6 mgfL and the free phenytoin was 4.2 mg/L. Because the calculated free fraction of 0.75 was not physiological, we remeasured the total and free phenytoin concentrations by HPLC (4). We obtained a nearly identical result for the free phenytoin concentration, but the total phenytoin concentration by HPLC was 24.2 mgfL, indicating an apparent recovery with the diluted Emit reagents of only 23%. The free fraction determined from the HPLC data was 0.17, a value consistent with the patient’s mild hypoalbuminemia (albumin 30 g/L) and azotemia (creatinine 53 mgfL). Further analysis of the patient’s serum revealed an LD activity of 9300 UIL and a lactate concentration of 21 minolJL. We hypothesized that the high U) activity could impair net NADH production, an effect that would be more pronounced when using diluted reagents. Reagent depletion by lactate can be ruled out: Even with a sample lactate concentration of 20 mmol/L, the NAD/Iactate ratio in the reaction mixture was >15. Therefore, other diluted Emit assays should be similarly affected. Because insufficient serum remained from the original specimen, in further experiments we used residual serum from four additional samples collected from the same patient over the next 12 h, with U) concentrations of 4800-9400 U/L. Known amounts of theophyllune (another analyte we assay with diluted Emit reagents) were added to the specimens. Analytical recovery when we used reagents diluted 20-fold averaged 36% (range 24-47%), values similar to those observed for phenytoin. The average recovery of theophylline when we used undiluted Emit reagents was 69% (range 60-78%). Thus, although reagent dilution contributed to the problem, undiluted reagents were also affected. The Emit method exhibited good recoveries for free phenytoin. Because (molecular mass 36.5 kDa) would have been excluded from the ultrafiltrate, we examined the effect of adding an U) inhibitor (sodium oxamate; Sigma Chemical Co., St. Louis, MO; final concentration 20 mmol/L) andreducing the dilution to fourfold (the U) concentrations of glucose 6-phosphate and NAD in reagent A remained 4.5 mmol/L). This resulted in an accept- able theophylline recovery of 89% (range 88-89%). Lack of additional specimen prevented testing these two modifications separately. Sodium oxa- mate did not affect the recoveries in specimens with normal LD activity. Examination of drug-supplemented sera from other patients with high U) activities by using diluted reagents without sodium oxaunate showed variable but generally more modest decreases in drug recovery than that observed in the index case (data not shown). The addition of sodium pyruvate (0.1 mmoJJL) to the sera further reduced recovery, but the decrease in recovery was stifi less than that seen in the index case. There was no apparent correlation between serum creatinine and drug recovery. We hypothesize that, in addition to increased serum activity of LD in our index case, increased concentrations of pyruvate or other ketoacids were also present. These putative interfering substances would generate NAD when reduced by a dehydrogenase, thus depleting the NADH generated in the Emit assay and producing falsely low results. We are not certain that U) is the only dehydrogenase involved. It may serve merely as a marker for tissue destruction and other enzymes might actually contribute to the problem. However, sodium oxamate apparently inhibited the activity of the enzyme(s) involved and minimized the problem. We conclude that sodium oxamate should be added to Emit reagents to minimize erroneous results in the presence of high concentrations of LD. References 1. Badcock NR, O’Reilly DA. False-positive EMIT#{174}-st ethanol screen with postmortem infant plasma [Tech Brief]. Clin Chem 1992;38:434. 2. Thede-Reynolds K, Johnson GF. False positive ethanol results by EMIT#{174} [Abstract]. Clin Chem 1993;39:1143. 3. Sung E, Neeley WE. A cost-effective system for performing therapeutic drug assays. I. Optimization of the theophylline assay. Clin Chem 1985;31:1210-5. 4. Roberts WL, Rainey PM. Interference in immunoassay measurements of total and 1598 CUNICAL CHEMISTRY, Vol. 40, No. 8, 1994 free phenytoin in uremic patients: a reappraisal. Clin Chem 1993;39:1872-7. William L. Roberts’ Florie S. Santos Petrie M Rainey Herbert Malkus Dept. Yale P.O. New of Lab. Med. Univ. School of Med. Box 208035 Haven, CT 06520-8035 Diagnostic Chemicals, Ltd.Jan Holinsky Oxford, CT 06478 ‘Author for correspondence. Analytical Performance of lmmulltem Assay of ThyroId-StImulatIng Hormone To the Editor: In the Immulite” (Cirrus Diagnostics, Diagnostics Products Corp., DPL Division, EURO/DPC, Glyn Rhonwy, LL55 4EL, UK) immunochemiluminometric assay (ICMA), thyroid-stimulating hormone (TSH; thyrotropin) is captured by a murine monoclonal antibody to TSH bound to a polystyrene bead (solid phase). The detector antibody, polyclonal goat antibody to TSH, is conjugated to alkaline phosphatase (ALP). After incubation, unbound conjugate is removed by a centrifugal wash, and the chemilumunescent substrate, a phosphate ester of adaniantyl dioxetane [3-(2’-spiroadamantane)-4methoxy-4-(3’-phosphoryloxy)-phenyl1,2-dioxetane], is added. In the presence of ALP this ester undergoes hydrolysis to form an unstable intermediate with the production of light. The light output, detected by a luminometer, is proportional to the concentration of TSH in the sample. The original description of the Immulite TSH method assessed the imprecision of the assay over the range 0.34 to 32.8 mIU/L (1). In contrast, the assay was designed for use at TSH concentrations two orders of magnitude lower than this, i.e., as a third-generation assay with a functional sensitivity (CV of <20% at 0.01 mIU/L) two orders of magnitude below that of typical firstgeneration TSH radioimmunoassays (2). In the present study, we have assessed the analytical performance of this assay over a lower range, using the functional sensitivity limit suggested by Nicoloff and Spencer (3). Immulite TSH assays were performed in singleton according to the manufacturer’s instructions. Results