Effects of Sodium Azide on the Quantitation of the Chemical Constituents of Serum

Transcription

1 Effects of Sodium Azide on the Quantitation of the Chemical Constituents of Serum Inhibition of Bilirubin and Cholesterol HASSAN KHAYAM-BASHI, PH.D., AND CHARLES SIMS, M.S. Khayam-Bashi, Hassan, and Sims, Charles: Effects of sodium azide on the quantitation of the chemical constituents of serum. Inhibition of bilirubin and cholesterol. Am J Clin Pathol 69: ,1978. Azide salts frequently are added as a preservative to biologic fluids and reagents, or are introduced into serum through accidental exposure, intoxication, or pharmaceuticals. Sodium azide can interfere with the quantitation of biochemical constituents in serum. Serum pools containing mg/dl ( fimoml) total bilirubin, -5.0 mg/dl ( jnmol/1) direct bilirubin, and mg/dl ( mmol/l) cholesterol were analyzed using the SMA 12/60. Sodium azide was added in concentrations of -1.0% ( mmol/l). Sodium azide in concentrations of % (13.6 mmol/l) or more reduced total and direct bilirubin values %. At concentrations above 0.5% (68 mmol/l), no bilirubin, or only a very small quantity, was measured. Sodium azide at concentrations above 5% (6.8 mmol/l) exerted a significant decreasing effect on serum cholesterol values. At % (13.6 mmol/l) or more, reductions in cholesterol values ranging from 30 to 85% were observed. These studies showed that sodium azide (%, 13.6 mmol/l, or more) in the serum can result in falsely low bilirubin or cholesterol values. (Key words: Sodium azide; Cholesterol; Total bilirubin; Direct bilirubin; SMA 12/60.) SODIUM AZIDE, an antibacterial agent with bacteriostatic properties, frequently is used at -% ( mmol/l) to preserve laboratory reagents and biological specimens, and its use at 0.5% w/v (68 mmol/l) to preserve ethanol in blood specimens has been recommended. 26 However, in addition to its potential safety hazard in the formation of explosive copper or lead azides, 28 sodium azide in the serum can influence the values obtained in laboratory tests for certain serum constituents and potentially can affect the accuracy of a diagnostic decision. Sodium azide is used agriculturally as a biodegradable herbicide and insecticide, has certain pharmaceutical uses, and appears as an environmental pollutant Received October 19, 1976; received revised manuscript March 16, 1977; accepted for publication March 16, Address reprint requests to Dr. Khayam-Bashi: Department of Laboratory Medicine, Clinical Laboratories, Building 100, San Francisco General Hospital, San Francisco, California Department of Laboratory Medicine, University of California, San Francisco, at the Clinical Laboratories, San Francisco General Hospital, San Francisco, California as a result of its use in rocket fuels and industrial processes. 7 Persons involved in the production or use of azide-containing substances may inhale sodium azide or absorb it through the skin. At a toxic level of azide in the serum (70 /ig/kg body weight), 22 these persons evidence symptoms of poisoning, such as tachycardia, hyperventilation, and hypotension. 22 Sodium azide can affect the physiologic processes, 7 can result in neuropathologic changes, 1516 and has metabolic effects. It binds to myoglobin and hemoglobin 1 and inhibits nuclear phosphorylation, 15 cytochrome oxidase, 15 protein synthesis, 15 ceruloplasmin activity, 27 and granulocytic function. 13 It also has been described as a mutagenic agent, 24 and is an activator of adenylate cyclase in the rat adipocyte plasma membrane. 20 A serum specimen may contain sodium azide as a result of a patient's environmental exposure or intoxication, or may be preserved with this compound after it is received in the laboratory. In either case, the presence of the compound in the serum can affect a variety of chemical reactions and interfere with the results of certain laboratory measurements. Although sodium azide does not affect such serum constituents as SGOT 21 and immunoglobulins, 2 there is recent evidence that it does interfere with the quantitation of other biochemical constituents in serum. Sodium azide interference has been documented in cholesterol measurements made by procedures that use reagents containing iron, 12 in the measurement of urea nitrogen by the diacetyl procedure, 3 and in the measurement of salicylates by Trinder's method. 19 This study was made to determine the effects of sodium azide on the chemical constituents of serum measured by the SMA 12/60. The results showed significant interference with cholesterol and bilirubin $00.75 American Society of Clinical Pathologists 405

2 406 KHAYAM-BASHI AND SIMS A.J.C.P. April 1978 (total and direct) measurements, which could lead to an erroneous interpretation of the values obtained. Materials and Methods Using an SMA 12/60 (Technicon, Terrytown, NY), the following automated procedures were performed: 1. Alkaline phosphatase (ALK), EC , by the method of Morgenstern and associates Calcium by the method of Kessler and Wolfman Cholesterol by the method of Holub and Galli 8 4. Inorganic phosphorus by the method of Hurst 9 and Kraml Lactate dehydrogenase (LDH), EC , and aspartate amino-transferase (glutamic acid oxalate transaminase; GOT), EC , by the method of Kessler and associates Total protein by the method of Skeggs and Hochstrasser Total bilirubin by the method of Gambino and Freda 5 8. Direct bilirubin by the method of Gambino and Schreiber 6 9. Uric acid by the method of Musser and Ortegoza Globulin by the method of Savory, Heintges and SobeF 11. Creatinine by the method of Chasson, Grady, and Stanley 4 Reagents for globuiin and cholesterol were obtained from Dow Diagnostics.* The p-nitrophenylphosphate (PNP) substrate for the alkaline phosphatase procedure was obtained from Searle Diagnostic, Inct All other reagents were obtained from Environmental Chemical Specialties. $ Preparation of Serum Pools The following serum pools were studied: 1) Commercially lyophilized quality control pools (Q, and Q 2 ) obtained from Hyland Division of Travenol Laboratories. 2) Two pools of patient sera from the SFGH Clinical Laboratory (Pool A and Pool B), and one (Pool H) obtained from a healthy individual, were * Dow Chemical Co. t Vallejo, Cal. t Placentia, Cal. Costa Mesa, Cal. Indianapolis, Ind. Table I. Effect of Sodium Azide on Serum Cholesterol Measurement mg/dl* Cholesterol in Serum i Containing Sodi um Azide: 25% 5% 0% 5% 0.50% 0.75% 1.00% Q, 175 Pool H (fresh) 218 Pool H (6 days old) 216 Pool B * For conversion to SI units (/Amol/I) for bilirubin, each value should be multiplied by Table 2. Effect of Sodium Azide on Bilirubin Measurement mg/dl* Bilirubin in Serum Containing Sodii jm Azide: 25% 5% 0% 5% 0.50% 0.75% 1.0% Q, Q,and Q 2 Q 2 Pool A Pool H (fresh) Pool H (6 days old) Direct Q, Q 2 Pool A Pool H (fresh) Pool H (6 days old) * For conversion to SI Units (/imol/1) for bilirubin, each value should be multiplied by 17.1.

3 Vol. 69 No. 4 9r-*-t POOL A B1LIRUBIN/CHOLESTEROL INHIBITION BY AZIDE 9r-« > J, z O Ol E u z I CO 3 o o 1 - AZIDE CONCENTRATION (% W/V) FIG. 1. Effects of increasing concentrations of sodium azide on total bilirubin. Two pools. A and Q 2, with bilirubin concentrations of 9 mg/dl (153.9 /Ainol/1) and 4.2 mg/dl (71.8 /xmol/1), were used. used. Pool A was made from samples with high direct bilirubin values. Pool B contained samples with high cholesterol values and was used for cholesterol assay. Pools A and H were used for bilirubin assays. The serum pools were treated with sodium azide in the following manner: A stock solution of 36% w/w (4.9 M) sodium azidel in water was prepared, and 1% (136 mmol/1) serum azide was prepared by mixing 85 ml of stock solution with 1 ml of a serum pool. A corresponding serum diluent (azide-free) control was prepared by mixing 85 ml water with another 1 ml portion of the serum pool. Varying proportions of stock 1% (136 mmol/1) serum azide were mixed with serum diluent to obtain decreasing concentrations of sodium azide (1-0%) for each serum pool. Serum samples were assayed on the day of preparation. Pool H was stored in the refrigerator at 7 C and reassayed after six days. Results Tables 1 and 2 show the effects of sodium azide on the quantitation of bilirubin and cholesterol. At and H Mallinkrodt, St. Louis, Mo. AZIDE CONCENTRATION (% W/V) FIG. 2. Effects of increasing concentrations of sodium azide on total (T.B.) and direct (D. B.) bilirubin concentrations. The serum pool contained 9.0 mg/dl (153.9 Aimol/1) total and 5.0 mg/dl (94.1 /j.mol/1) direct bilirubin. 5%(13.6and 34 mmol/1) serum azide concentrations, cholesterol values were depressed 30 and 60%, respectively (Table 1). The extent of depression depended upon the azide concentration and was independent of cholesterol concentration. The effects of sodium azide on total bilirubin for several specimens ranging from to 9.0 mg/dl ( /xmol/1) bilirubin are shown in Table 2. Figure 1 shows this inhibition for two pools (Pools A and Q 2 ) and indicates that at sodium azide concentration above % (13.6 mmol/1), a significant and pronounced inhibition occurred. Most total and direct bilirubin values were depressed to zero at 5% (34 mmol/1) sodium azide, although the sample with a 9.0 mg/dl (153.9 /xmol/1) total bilirubin value was depressed to mg/dl (13.7 /imol/l) at this concentration (Pool A, Table 2; Fig. 2). At % (13.6 mmol/1) and higher sodium azide concentrations, the measured direct bilirubin values were higher than total bilirubin values. The sample with 5.0 mg/dl (85.5 /xmol/ 1) direct bilirubin was depressed only 46% to 2.7 mg/dl (46.2 /i.mol/1) at 5% (34 mmol/1) sodium azide, and still measured (13.7 ptmol/1) at 1.0% (136 mmol/1) sodium azide (Fig. 2).

4 408 KHAYAM-BASHI AND SIMS A.J.C.P.. April 1978 Alkaline phosphatase values were slightly depressed (6%) at 5% (34 mmol/l) sodium azide. Samples with significant alkaline phosphatase activity appeared to be depressed more than those with low or minimal enzymatic activities, which might indicate that certain alkaline phosphatase isoenzymes were affected more than others. LDH values were decreased by about 5 IU at 5% (34 mmol/l) sodium azide. No LDH value was depressed more than 8%, even at 1% (136 mmol/l) sodium azide. Calcium values also may be affected by azide; some of the samples showed a slight increase of 1-3% in calcium content at 5% (34 mmol/l) sodium azide and a slightly greater increase at higher sodium azide concentrations. None of these changes was sigr nificant. Sodium azide appeared to have no effect on any of the other constituents measured. Discussion The data clearly show that sodium azide radically depresses cholesterol and bilirubin values determined by the SMA 12/60 procedures described here. LDH and alkaline phosphatase values also were slightly, but not significantly, affected, being depressed by not more than 6% at sodium azide concentrations of less than 0.5% (68 mmol/l). The values for the other SMA 12/60 tests performed were not affected by sodium azide in serum using these procedures. An in-depth study of the effect of sodium azide on cholesterol 12 showed that sodium azide affected those methods that used non-extracted serum and a reagent mixture containing iron. The results of the present study indicated that the depression of cholesterol values was highly significant when a sodium azide concentration of more than % (13.6 mmol/l) was used in conjunction with the SMA 12/60 method of Holub and Galli, 8 which utilizes iron-containing reagents. Our preliminary data showed that triglyceride values obtained using an enzymatic procedure were not affected by the sodium azide concentrations used in these studies (-1.0%, mmol/l). This finding indicates that in hyperlipemic conditions serious diagnostic errors can occur when a specimen shows high triglyceride and artifactually lowered cholesterol values. The effect of sodium azide on direct and total bilirubin is dramatic. As shown in Table 2 and Figures 1 and 2, at % (13.6 mmol/l) sodium azide, Pool A, which had high total bilirubin and high direct bilirubin in a ratio of 9.0/5.0, was affected such that it had a total bilirubin value of 2.9 mg/dl (49.6 /imol/1) and direct bilirubin of 4.2 mg/dl (71.8 fimo\j\). At higher azide concentrations, this apparent reversal of the ratio became even more striking, so that, at 0.5 and 1.0% (68 and 136 mmol/l), ratios of /1.5 and / were observed (Fig. 2). The significant effects of sodium azide on biochemical measurement of bilirubin and cholesterol, and its lesser effects on the quantitation of other serum constituents, suggest that indiscriminate use of sodium azide must be avoided, especially for the measurement of cholesterol (using iron-containing methodologies 12 ) and bilirubin (common procedures used in this study 5,6 ). The drastically and artifactually reduced total bilirubin values obtained with sodium azide, even at concentrations as low as % (13.6 mmol/l), are particularly alarming when they have been obtained in critical specimens, especially when administration of a blood transfusion to a newborn may be indicated. When it is suspected that sodium azide is present, either alternative methods that are not affected by sodium azide should be used, or the azide concentration in the serum should be determined, and if possible, appropriate corrections should be made. References 1. Alben JO, Fager LW: Infrared studies of azide bound to myoglobin and hemoglobin: Temperature dependence and ionicity. Biochemistry II, , Campbell DH, Garvey JS, Cremer NE, et al: Methods in Immunology. Second edition. New York, W. A. Benjamin, 1970, p30 3. Caraway WT, Kammeyer CW: Chemical interference by drugs and other substances with clinical laboratory test procedures. Clin Chim Acta 41: , Chasson AL, Grady HT, Stanley MA: Determination of creatinine by means of automatic chemical analysis. Am J Clin Pathol 35:83-88, Gambino SR, Freda VJ: The measurement of amniotic fluid bilirubin by the method of Jendrassik and Grof. Am J Clin Pathol 46: , Gambino SR, Schreiber H: The measurement and fractionation of bilirubin on the AutoAnalyzer by the method of Jendrassik and Grof, Automation in Analytical Chemistry. Technicon Symposia (Technicon Corp, Tarrytown, N. Y.), Graham JDP: Actions of sodium azide. J Pharmacol 4:1-6, Holub WR, Galli FA: Automated direct method for measurement of serum cholesterol with the use of primary standards and a stable reagent. Clin Chem 18: , Hurst RO: The determination of nucleotide phosphorus with a stannous chloride-hydrazine sulphate reagent. Can J Biochem 42: , Kessler G, Rush RL, Leon L, et al: Automated 340 measurement of SGOT, SGPT, and LDH. Advances in Automated Analysis, Volume 1. Technicon Int Cong, Miami, Thurman Associates (Miami), 1971, pp Kessler G, Wolfman M: An automated procedure for the determination of calcium and phosphorus. Clin Chem 10: , Khayam-Bashi H, Sims C, Parekh AC: Interference of sodium azide with the quantitation of serum cholesterol: A comparative study. Clin Chim Acta 66:69-75, Koch C: Effect of sodium azide upon normal and pathological granulocyte function. Acta Pathol Microbiol Scand B, 82: , 1974

5 Vol. 69 No. 4 BILIRUB1N/CHOLESTEROL INHIBITION BY AZIDE Kraml M: A semi-automated determination of phospholipids. Clin Chim Acta 13: , Mettler FA: Neuropathological effects of sodium azide administration in primates. Fed Proc 31: , Mettler FA, Sax DA: Cerebellar cortical degeneration due to acute azide poisoning. Brain 95: , Morgenstern S, Kessler G, Auerbach J, et al: An automated p-nitrophenylphosphate serum alkaline phosphatase procedure for the AutoAnalyzer. Clin Chem 11: , Musser AW, Ortegoza C: Automated determination of uric acid by the hydrazine method. Tech Bull Reg Med Tech 36:21-25, Queen CA, Frings CS: Effect of sodium azide on Trinder's method for the determination of salicylate. Clin Chim Acta 45:307, Rahmanian M, Jarrett L: Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide. Biochem Biophys Res Commun 61: , Rej R, Vanderiinde RE: Azide as a preservative in assays of aspartate aminotransferase activity. Clin Chem 21: , Roberts RJ, Simmons A, Barrett DA, II: Accidental exposure to sodium azide. Am J Clin Pathol 61: , Savory J, Heintges MG, Sobel RE: Automated procedure for simultaneously measuring total globulin and total protein in serum. Clin Chem 17: , Sideris EG, Argyrakis M: Chemical alterations induced in DNA and DNA components by the mutagenic agent azide. Biochim Biophys Acta 366: , Skeggs LT, Jr, Hochstrasser H: Multiple automatic sequential analyzer. Clin Chem 10: , Smalldon KW: Ethanol oxidation by human erythrocytes. Nature 245: , Smith BSW, Wright H: Improved manual and automated procedures for estimation of ceruloplasmin oxidase activity. Clin Chim Acta 50: , Wear JO: Azide hazards with automatic blood cell counters. J Chem Educ 52:A23-A25, 1975