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DO. Use of specimen turnaround time as a component of laboratory quality-a comparison of clinician expectations with laboratory performance. Am J Cliii Pathol 1989;92:613-8. 5. Henderson AR. The priority test request form: a method for improving communication between the physician and the emergency clinical biochemistry laboratory. J Cliii Pathol 1979;32: 97-9. 6. Henderson AR. The test request communication between the physician form: a neglected route for and the clinical chemist? J Cliii Pathol 1982;35:986-98. 7. Valenstein PN, Emancipator K. Sensitivity, specificity, and reproducibility of four measures of laboratory turnaround time. Am J Cliii Pathol 1989;91:452-7. 8. Gardner MJ, Gardner SB, Winter PD. Confidence interval analysis microcomputer program. London: British Medical Journal, 1989:77pp. 9. Hilborne LH, Oye RK, McArdle JE, Repinaki JA, Rodgerson DO. Evaluation of stat and routine turnaround times as a componentof laboratoryquality. Am J Clin Pathol 1989;91:331-5. 10. Howanitz PJ, Steindel SJ. Intralaboratory performance and laboratorians’ expectations forstat turnaround times. Arch Pathol Lab Med 1991;115:977-83. 11. Barnett RN, MclverDD, Gorton WL. The medical usefulness of stat tests. Am J Clin Pathol 1978;69:520-4. 12. Howanitz PJ, Steindel SJ, Cembrowski GS, Long TA. Emergency department stat test turnaround times. Arch Pathol Lab Med 1992;116:122-8. 13. McConnell TS, Writtenberry-Loy C. Whither waiting turnaround times of laboratory tests for emergency room patients. Lab Med 1983;14:644-7. 14. Donabedian A. Explorations in quality assessment and monitoring. Vol. 1. The definition of quality and approaches to its assessment. Ann Arbor: Health Administration Press, 1980:163 pp. 15. Juran JM. Juran on quality by design. New York: The Free Press, 1992:538 pp. 16. Selker HP, Beshsnsky JR, Pauker SG, Kassirer JP. The epidemiology of delays in a teaching hospital. The development and use of a tool that detects unnecessary hospital days. Med Care 1989;27:112-29. 17. Valenstein PN. Turnaround time-can we satisr clinicians’ demands for faster service? Should we try? [Editorial]. Am J Cliii Pathol 1989;92:705-6. CUN. CHEM. 39/6, 1059-1063 (1993) Glutamine Stability in BiologicalTissues Evaluated by Fluorometric Analysis V. Bruce Grossie, Jr.,”3 John Yick,’ Mark Alpeter,’ Thomas C. Welbourne,2 and David M. 0th’ quantity of plasma (25 L) or tissue (200 mg) and is a Although glutamine has been considered unstable during convenient method for quantifying this important amino storage and therefore difficult to quantitate, recent results suggest this amino acid is stable at low pH ranges. We acid. evaluated the stability of glutamine in plasma and tissue extracts, using fluorometric analysis. The measured conIndexing Terms: amino acids sample handling centration of glutamine detected varied linearly up to 0.8 mmol/L for the aqueous solution (r2 = 98.7, P = 0.0001) Glutainine is an important amino acid for the nutriwith a mean (±SD) coefficient of variation of 2.41% ± tional regimens of catabolic and cancer patients (1-3). 0.79%. When glutamine was dissolved in 50 g/L trichloManipulation of the amino acid composition of total roacetic acid (TCA), the values were essentially unalparenteral nutrition formulas for catabolic and tumortered. Glutamine in an aqueous solution and stored at bearing hosts has received considerable attention in the -70#{176}C was stable for at least 16 days; glutamine in TCA past few years (1-11). Glutamine is the most abundant was stable for 6-8 days, then decreased to a concentracirculating free amino acid and in intracellular pools is tion significantly lower than that of the aqueous solution. a precursor for amino acid, protein, and nucleotide The expected and observed concentrations in plasma synthesis and is required for ammonia genesis by the were equal (r2 = 0.99975) for increasing amounts of kidney (12). added glutamine. Glutamine concentrations in plasma Glutamine has previously been considered to be unwere stable for >1 year when stored at -70 #{176}C. The stable in aqueous solutions when subjected to heat (12, glutamine of a transplantable rat sarcoma and a normal 13). Gilbert et al. (14) presented results showing that rat liver could be extracted with 50 g/L TCA with high the concentration of anions such as phosphate adversely efficiency (88.6% ± 1.9% and 90.2% ± 0.04%, respecaffected glutaunine stability. Herskowitz et al. (15), tively); the extracted glutamine is stable in TCA for at least however, reported that glutamine concentration is sta7 days without neutralization when stored at -70 #{176}C.ble at refrigerator temperatures (5#{176}C, pH 6.2) for 2 Fluorometric analysis of glutamine required only a small days; stored at -20#{176}C (pH 6.2), the concentration of glutamine was stable for 3-7 days. Shih (16) demonstrated that the stability of glutamine at 37#{176}C for 24 h 1Department of General Surgery, Box 106, The University of was 100% at pH 3 but decreased with increasing pH to Texas M.D. Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030. -35% at pH 10. Rosenblum (17) reported that gluta2Department of Physiology and Biophysics, LSU Medical Cenmine remained stable when prepared in citrate or aqueter, Shreveport, LA. ous solution and deproteinized plasma. ‘Author for correspondence. No study has evaluated the stability of glutamine in Received July 13, 1992; accepted January 18, 1993. CUNICAL CHEMISTRY, Vol.39, No. 6, 1993 1059 tissue extracts, however, even though many articles concerning the importance of this amino acid have been published. The purpose of this study was to evaluate the stability of glut.amine in plasma and tissue extracts by using a fluorometric analysis for glutamine and gluta- B represent the results for glutainine, glutamate, stan- dard, and the blank, respectively. (R2GLN-R1GLN)-(R2B-R1B) f Glutamine = mate. 1 (R2ST-R1STD) 1 Materials and Methods x STD concentrationJ Procedures Assay. L-Glutamine (Sigma Chemical Co., St. Louis, MO) was assayed by a modification of the procedure of Lund (18), which measures the change in the fluorescence of NADH (Sigma Chemical Co.). The procedure is detailed in Table 1. The glutamine in the plasma or tissue extract was converted to glutamate by glutaminase (Grade V; Sigma Chemical Co.). The resulting glutamate was then assayed by measuring the NADH formed by the conversion of glutamate to a-ketoglutarate and ammonia by glutamate dehydrogenase (Boehringer Mannheim Corp., Indianapolis, IN). The NADH was measured with a Perkin-Elmer LS-50 Luminescence Spectrometer (Perkin-Elmer Corp., Stafford, TX) at an excitationwavelength of 342 nm (5-nm slit width) and an emissions wavelength of 459 nm (20-nm slit width). Calculations. The calculations for glutamine and glutainate in plasma (mo]/L) and tissues (mol/g wet tissue) were similar to those by Lund (18), where Ri and R2 represent the fluorescence values from the different stages of the assay (Table 1), and GLN, GLU, STD, and Table 1. Procedure for Spectrofluorometric Glutamate -glutamate 1(R2GLU-R1GLU)-(R2B-R1B) ST = L (R2 D-R1STD) x STD concentration Standard curve. Initially, glutamine was dissolved in sterile water or 50 g/L trichloroacetic acid (TCA) solution and assayed as shown in Table 1. Each concentration was assayed in triplicate, the coefficient of variation (CV) for each concentration was calculated as described by Zar (19), and the mean CV was then calculated from the CVs from the seven concentrations. Preliminary results demonstrated that 50 gIL TCA was the best acid for tissue extraction. The effect of 50 g/L TCA on the fluorometric assay and standard curve of glutamine was therefore compared with results for an aqueous solution. Stability: aqueous vs 50 gIL TCA Standard concentrations of glutamine were prepared in sterile water (n = 5) or 50 g/L TCA (n = 5). One aliquot of each solution Analysis for Glutamine and Glutamate In Plasma and Tissues Added to essay Reaction cemponsnt 2 3 4 5 6 Part II 1 2 3 4 5 6 7 8 9 Glutamlns Acetate buffer (1.0 mmol/L, pH 4.9), L Sample, L Standard,L Sterilewater, L Glutaminase,#{176} L Incubate(37 ‘C), mm Cold TCA (50 g/L), &L Centrifuge, mm Pyrophosphatebuffer (26 g/L, pH 8.6), L Samplefrompartl,uL NAD (20 g/L 30 mmol/L), L Glutamate 25 55 25 25 Standard 25 Blank 25 25 30 25 30 60 60 180 180 30 60 180 10 10 10 10 2000 140 2000 2000 140 2000 140 40 40 8 8 8 45 45 45 40 Mbctwuce Measure fluorescence three times and averagereading(Ri) GLDH (20 U/mg),d&L 8 Mix twice Incubateat room temperature (covered), mm 45 Measure fluorescence(meanof three readings:R2) OC 140 40 60 180 a The assay l done in two parts. The initial reactionin part lis donein 16 x 100 mm glass tubes. In the second part, the pyrophosphate buffer, sample from part I, and NAD are mixed directly In a precision cell (NSGPrecision Cells, Farmingdale,NY). Fluorescence is then read In these cells by thefluorometer. 5Glutamlnase (1-glutamineaminohydrolase; EC 3.5.1.2) from Escherichia coIl, 10 U in 1.0 mL of acetate buffer. Additionof glutaminaseafterthe Incubation(1-4)and coldTCA (1-5)was demonstratedto have noeffectonthe results; giutaminasewas thereforenot added to thustobe. d Glutamate dehydrogenase I.-glutamate:NAD(P) oxidoreductase(EC 1.4.1.3)). TCA, trichioroaceticacid. 1060 CLINICAL CHEMISTRY, Vol. 39, No. 6, 1993 was assayed immediately (day 0). The remainder of each solution was then separated, stored at -70 #{176}C, and assayed at various intervals until a significant decrease in the glutamine concentration was observed. Biological Samples Plasma. Blood from a healthy evaluate the assay in plasma. volunteer was used to Increasing glutamine concentrations were added to aliquots of plasma and assayed immediately. Fresh blood from three healthy volunteers was drawn into heparini.zed tubes and the plasma separated by centrifugation at 4#{176}C; all procedures were done with the tubes on ice. Glutamine and glutamate were assayed immediately. Aliquots of each plasma sample were stored at -70 #{176}C and assayed at intervals as indicated in the text. Samples were kept frozen until the respective time of assay. Tissues. Lund (18) suggested that deproteinization of plasma with perchloric acid should be followed by immediate neutralization with potassium hydroxide; Se- bolt and Weber (20) and Quesada et al. (21) utilized this concept for tissue extractions. Results reported by Shih (16), however, suggested that glutamine was more stable at pH <2. The stability decreased as pH increased, and the acid hydrolysis product-pyroglutamic acidbegan to appear. Initially, homogenization with 40 gIL sulfosalicylic acid (SSA; Sigma Chemical Co.) was used because of its successful utilization for extraction of tissues for determination of other amino acids and polyamines for HPLC analysis. The fluorescence background was high, however, and we attempted no stability studies with samples so treated. We then tried homogenizing tissues in 50 g/L cold TCA (6 mLfg tissue), using a Polytron tissue homogenizer (Brinkmann Instruments, Westbuiy, NY). The homogenate was then centrifuged at 100 000 x g for 45 mm at 4#{176}C, and the clear supernate was assayed as described in Table 1. This procedure was repeated with the initial precipitate and then repeated a second time; the glutainine concentration in the clear supernate was determined to evaluate the recovery of glutamine 50 40 >.. 0 30 0 20 Cs Cs 10 - ever, the glutamine concentration (91.12% ± 1.72% of Aqueous 0 5%TCA 0 0.0 0.5 1.0 1.5 Glutamine concentration (mmol/L) Fig. 1. Unearityofglutammneconcentrationwhenformulatedin sterile water or a 50 g/L TCA solution on detectionby fluoromettic analysis The detection of glutammnewas linear up to 0.8 mmol/L for the aqueous solution (r2 = 98.7, p = 0.0001) wIth a mean ± SD CVof 2.41% ± 0.79%. Dissolvingglutamine in 50 g/L TCA had no effect on assay values the initial concentration) was significantly (P = 0.0009) lower than that of the respective aqueous solution. The expected and observed concentrations in plasma when increasing amounts of glutamine were added are shown in Figure 3. Glutamine assayed in plasma was equal to that expected from the sum of the original plasma concentration and the amount added (r2 = 0.99975). The stability of glutammne in plasma from three normal male volunteers was evaluated (data not shown). Although the initial concentration varied among individuals (785, 636, and 809 A.moI/L), the glutainmne concentrations measured in stored plasma (as a percentage of the initial value) were stable for >1 year at -70#{176}C (data not shown). The recovery of glutamine from tumor (n = 8) and liver (n = 3) tissue after extraction with 50 g/L TCA (6 mL/g) was evaluated as well as the effect of storage of C 0 125 Aque -0-rCA C) ...0 of 2.41% ± 0.79%. Assaying glutamine dissolved in 50 g/L TCA solution did not change the results. The effect of storage conditions (time and diluent) on the stabifity of the solutions (as a percentage of day 0 concentration) is shown in Figure 2. Glutamine formulated as an aqueous solution and stored at -70 ‘C was stablefor at least 16 days (97.94% ± 2.14% of the initial concentration). When formulated in the TCA solution, the glutamine concentration was stable for 6(98.64% ± 1.86%) and 8 days (96.34% ± 2.72%). By day 16, how- a LJ in 50 g/L TCA. Results The effect of increasing glutamine concentrations in aqueous and 50 g/L TCA solutions on the relative intensity is shown in Figure 1. The amount of glutamine detected was linear up to 0.8 mmoIJL for the aqueous solution (r2 = 0.987, P = 0.000 1) with a mean (± SD) CV I1 - to 100 1 wE ace :i1 ‘ ab 75 0 10 20 Days of storage FIg. 2. Effectof storagetime on the stabilityof glutammne formulated in sterilewater (n = 5) or 50 g/L TCA (n = 5) The respectivesolutions wereformulatedandassayedimmediatelyor stored at -70 ‘C for the indicated number of days. The percentage of Initial concentrationremainingat eachtimepointwascalculated;the mean ± SD Is reported.(a) The glutamlne concentration of the aqueous solution differs significantly (P = 0.015) fromthat in TCA solution on day 14 afterformulation. ( The glutammne concentrationofthe TCA solutiondifferssignificantlyfrom thatin the aqueous solutionon day 16 after formulation (P = 0.0009) andfrom the initial glutamineconcentration formulatedin TCA (P = 0.0011). The concentrationof glutamine formulated in sterile water at day 16 was equal to that at day 0(P= 0.13) CLINICALCHEMISTRY, Vol. 39, No. 6, 1993 1061 tered by Khan et al. (22), who demonstrated that the absorbance of SSA was significant at 340 nm, the maximum wavelength of absorbance for NADH. These authors demonstrated that, although this problem could 1.5 -J 0#{176} 1.0 Cs 0“-C 0.5 too 00 0.0 0.0 1.0 0.5 Expected 1.5 Iutamlne concentration (mmol/L) Fig.. 3. EffIciencyof the determination of glutamine in plasma Increasing concentrations of glutamine were added to a plasmasample of known glutamineconcentration and assayed spectrofiuoiometrIcalIy. The glutamine assayed Inplasma was 97% of that expected (r2 = 0.99975) the supernate at -70 ‘C for 7 days; the results are shown in Table 2. The mean recovery of glutamine from the first of three extractions was 88.6 1% from tumor and 90.20% from liver. Glut.amine was stable at -70#{176}C in the tumor tissue extract for at least 7 days. The pH of the initial supernate from the homogenization of liver and tumor was 1.53. Discussion The importance of glutamine as a nutrient for parenteral and enteral formulas has been shown repeatedly (1-3). Our results (4) show that when arginine in total parenteral nutrition is replaced with ornithine the plasma concentrations of glutamine will increase; replacing arginmne with citrulline has no effect. The ability to efficiently determine glut.ainine concentrations in plasma and tissues is, therefore, important to the understanding of the effects of nutritional manipulations. Our original objective was to evaluate the effect of different acids for tissue homogenization and to determine the best neutralization method for efficient analysis of glut.amine in plasma and tissues. For tissues, we first used a 40 gIL SSA solution because of its wide use in the determination of other amino acids. However, SSA resulted in a high fluorescence background, and its use was discontinued. This problem was also encoun- be reduced by increasing the measurement wavelength to 355 nm, the sensitivity for detection of NADH was also decreased. The spectrum for SSA was not established for our conditions, but the problem of decreased sensitivity would be critical. Given the interference of SSA at 340 nm and the probable decreased sensitivity for glutamine at 355 nm, we used TCA in subsequent experiments for tissue extraction. An important observation was that neutralization after TCA extraction of tissues was not necessary. This significantly decreased the processing time for each sample and also reduced the overall error contributed by dilution. The pH of the supernate from the TCA extraction of liver and tumor was lower than previously reported, suggesting that glutamine is stable under acidic conditions. The present results are in agreement with Herskowitz et al. (15) and Shih (16), who showed that glutamine is stable under acidic conditions for 5 days if stored at -70 ‘C. Khan et al. (22) demonstrated that glutamine was stable for -8 days after extraction of plasma with SSA (40 g/L). The plasma concentrations for male volunteers as determined by our assay, as well as the accuracy of the assay, correspond well with those reported for other methods of glutamine analysis (15, 16, 23). Our results suggest that glut.amine in plasma is stable for at least 12 months when stored at -70 ‘C. The glutamine concentrations in rat liver (Table 2) are lower than those reported by Sebolt and Weber (20), whereas the concentrations in the sarcoma (Table 2) correspond with the values reported (20) for medium to rapidly growing hepatomas. The sarcoma used was a transplantable tumor, which grows exponentially (24). The results in this report, therefore, suggest that glutamine is stable when appropriate, well-defined storage conditions are used. Tissues may be extracted for glutamine analysis with 50 g/L TCA without subsequent neutralization. The spectrofluorometric analysis of glutamine allows analysis of a small amount of plasma (25 L) and tissue (200 mg) with acceptable accuracy. Table 2. EfficIency of ExtractIonof Glutamlne from Rat Tumor and Uver TlssuV Mean ± SD after extraction s-i &mol/gtissue Tumor (n = 8) 1.37 ± 0.44 (1.38 ± 0.42)0 Liver (n = 3) 3.41 ± 0.20 References p-i Sot total 88.6 (89.4 ± ± 1.9 2.2) 90.2 ± 0.4 pmoi/g tissue S of total 0.18 ± 0.05 (0.17 ± 0.06) 11.4 0.37 ± 0.40 ± 1.9 (10.7 ± 2.2) 9.8 ± 0.4 OnIy results for the initial supemate (S-i) and pellet (P-i) are shown. Subsequent extractions of pellet yielded negligible glutamine concentration. 0The concentrations assayed after 7-10 daysof storage at -70#{176}C are listed in parentheses. 1062 CLINICAL CHEMISTRY, Vol. 39, No. 6, 1993 Supported by grants from Clintek Technologies, DeerfIeld, IL, and the Department of Health and Human Services, NCI CA34465. 1. Klimberg VS, Souba WW, Salloum EM, Plumley DA, Cohen FS, Dolson DJ, et al. Glutamine-enriched diets support muscle glutamine metabolism without stimulating tumor growth. J Surg Res 1990;48:319-23. 2. Chance ‘NT, Can L, Fischer JE. Response of tumor and host to hyperaliment.ation and antiglutamine treatments. J Parent Ent Nutr 1990;14:122-8. 3. Hammarqvist F, Wernerman J, All R, von der Decken A, Vinnars E. Mdition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall of protein synthesis,and improves nitrogen balance. Ann Surg 1989;209:455-61. 4. Grossie VE Jr, Nishioka K, Ajani JA, Ota DM. Substituting ornithine for arginine in total parenteral nutrition eliminates enhanced tumor growth. J Surg Oncol 1992;50:161-7. 5. Tachabana K, Mukai K, Moriguchi S, Takaina S, Kishino K. Evaluation of the effect of arginine-enrichedamino acid solutions on tumor growth. J Parent Ent Nutr 1985;9:428-34. 6. GosekiN, Endo M, OnoderaT, Kosaki G. Influenceof L-methionine-deprived total parenteral nutrition on the tumor tissue and plasma amino acids fraction and the host metabolism:experimental study with Sate lung carcinoma-bearing rats. Tohoku J Exp Med 1989;157:251-60. 7. Martensson J, Larsson J, Schildt B. Metabolic effect of amino acid solutions in severelyburned patients: with emphasison sulfur amino acid metabolism and protein breakdown. J Trauma 1985; 25:427-32. 8. Tayek JA, Bistrian BR, Hehir DJ, Martin R, Moldawer LL, Blackburn GL. Improved protein kinetics and albumin synthesis by branched-chain amino acid-enriched total parenteral nutrition in cancer cachexia. A prospective randomized crossover trial. Cancer 1986;58:147-57. 9. Mon E, Hasebe M, Kovayashi K Effect of total parenteral nutrition enriched in branched-chain amino acids on metabolite levels in septic rats. Metabolism 1988;37:824-30. 10. Hunter DC, Weintraub M, Blackburn GL, Bistrian BR. Branched chain amino acidsas the protein component of parenteral nutrition in cancer cachexia. Br J Surg 1989;76:149-53. 11. Mon E, Hasebe M, Kobavashi K, Suzuki H. Immediate stimulation of protein metabolism in burned rats by total parenteral nutrition enriched in branched chain amino acids.J Parent Ent Nutr 1989;13:484-9. 12. Smith RJ. Glutamine metabolism and its physiologicimportance. J Parent Ent Nutr 199o;14:40S-448. 13. Greenstein JP, Winitz M. Glutamic acid and glutamine. In: Chemistry ofthe amino acids. New York: John Wiley & Sons,Inc., 1961:1933-4. 14. Gilbert JB, Price YE, Greenstein JP. Effect of anions on the nonenzymatic desanudation of glutamine. J Biol Chem 1949;180: 209-48. 15. Herskowitz K, Baumgartner TG, Austgen TB., Chen MK, Souba WW. Stability and sterility of glutamine in solution [Abstract]. J Parent Ent Nutr 1990;14:198. 16. Shih FF. Analysis of glutamine, glutamic acid, and pyroglutamic acid in protein hydrolysates by high-performance liquid chromatography.J Chromatogr 1985;322:248-56. 17. RosenblumR. Stability of glutamine in vitro. Proc Soc Exp Biol Med 1965;119:763-5. 18. Lund P. UV-method with glutaminase and glutamate dehydrogenase.In: BergmeyerHIJ, ed. Methods of enzymatic analysis, Vol. 8, 3rd ed. New York: Academic Press, 1974:357-64. 19. Zar JR. Measure of dispersion and variability. In: Biostatistical analysis, 2nd ed. Princeton, NJ: Prentice-Hall, 1984:31-2. 20. Sebolt JS, Weber G. Negative correlation of L-glutamine concentrations with proliferation rate in rat hepatomas. Life Sci 1984;34:301-6. 21. Quesada AR, Medina MA, Marques J, Sanchez-Jimenez FM, de Castro JN. Contribution of host tissues to circulating glutamine in mice inoculated with Erlich ascites tumor cells. Cancer Res 1988;48:1551-3. 22. Khan K, Blaak E, Ella M. Quantifying intermediate metabolites in whole blood after a simple deproteinization step with sulfosalicylic acid. Clin Chem 1991;37:728-33. 23. Stahl A, Frich A, Imier M, Schlienger J-L Enzymatic microassay for bloodglutamine. Clin Chem 1978;24:1730-3. 24. Grossie YB, Nishioka K, Ota DM, Martin RG. Relationship of erythrocyte polyamines and the growth rate of transplantable tumors in the rat. Cancer Res 1986;46:3463.-8. CUNICAL CHEMISTRY, Vol. 39, No. 6, 1993 1063