For most of the tests performed on the Ektachem plasma. CLIN. CHEM. 35/1 ..... Coloscreen VPI Test Kit Evaluated for Detection of Fecal Occult Blood. Anthony J. ... guaiac (impregnated into test cards) as the pseudoperoxi- dase substrate and ...
assays for a few hours would not misguide the physician in the diagnosis of disease. We also examined the stability of creatimne and found it to be unchanged for at least five days. Finally, a word of caution for those who use the pierceable caps for the purpose of reducing the rate of sample evaporation during analysis. These caps should be used for their intended purpose only and not for long storage of specimens (most laboratories save specimens for a few days in case an additional test or verification of a questionable result is requested), because we found a 3% per day lossof water from specimens capped with pierceable caps and kept at 4#{176}C. For most of the tests performed on the Ektachem plasma
is indistinguishable from serum. Only for amylase, AST, and perhaps potassium, can plasma and serum not be used interchangeably. References 1. Carothers JE, Kurtz NM, Lemann Jr J. Error introduced by specimen handling before determination of inorganic phosphate concentrations in plasma and serum.Clin Chem 1976;22:1909-11. 2. Berg JD, Romano G, Bayley NF, Buckley BM. Heparin interferes with aspartate aminotransferase activity determination in the Ektachem 700. Clin Chem 1988;34:174. 3. Weissman N, Pillegi VJ. Inorganic ions.In: Henry RJ, Cannon DC, Winkelman JW, eds.Clinical chemistry, principlesand technice. Hagerstown, MD: Harper and Row, 1974:645-6.
CLIN. CHEM. 35/1, 153-156 (1989)
Evaluationof the IFCC Reference Method for Alanine Aminotransferase:SpuriousBlank ALT Activitydue to Contaminationof D-Alaninewith L-Alanine,and Recommendationsfor a Correction Anthony 0. Okorodudu, Peter R. Pefletler,Andre A. Valcour, George N. Bowers, Jr., and Robert B. McComb1
The International Federation of Clinical Chemistry (IFCC) reference method for alanine aminotransferase (ALT, EC 2.6.1.2) requires four different reactions-A, B, C,
and D-to obtain the corrected ALT activity (j).2 Reactions A and B measure the overall and reagent blank reaction rates with talanine as substrate, with and without sample, respectively. Reactions C and D are used to measure the specimen blank and the reagent blank activities by substituting the inactive n-alamne isomer for the active L-alanine (2). The blank-corrected ALT activity (UIL) is then calculated as [(A B) (C D)]. The use of this ri-alanine in reactions C and D presumably allows for maintaining nearidentical chemical properties for the two reaction mixtures and their respective blanks. The WCC ALT procedure noted the possibility that Lalanine may contaminate the r-alanine, leading to overcorrection for the ALT activity (1). To avoid this error, a procedure for checking the presence of i-alanine in the nalanine reaction mixture is recommended in the IFCC ALT procedure. This checking procedure, which is based on the use of ALT from porcine skeletal muscle, is somewhat cumbersome regarding the adjustment of the ALT concentrations and the dilution ratios of the substrate needed to bring the reaction to completion within the limits of absorbance change. Here we re-address the use of n-alanine in the IFCC ALT procedure in relation to contamination with i.alanine. First, we quantified the L-alanine contamination of n-alanine in five commercial lots of D-alanine preparations, using Iamino acid oxidase/peroxidase (L-AAOJPOD) coupled reaction systems, in which hydrogen peroxide generated in the L-AAO reaction is followed by an oxidative coupling catalyzed by horseradish POD to produce a red product (3). Next, we compared these results with the IFCC reference procedure for checking for the presence of L-
Clinical Chemistry Division, Department of Pathology,Hartford Hospital,Hartford, CT 06115. ‘Address correspondence to this author. ReceivedAugust 26, 1988; acceptedOctober 24, 1988.
2Nonstandard abbreviations: IFCC, International Federation Clinical Chemistry; ALT, alanine aminotransferase;L-AAO, amino acid oxidase; POD, peroxidase; 4-AAP, 4-aminoantipyrine; and HDBS, 2-hydroxy-3,5-dichlorobenzene sulfonicacid.
During an evaluation of the IFCC reference method for alanine aminotransferase (ALT, EC 2.6.1.2), we noted that the specimen blank activity reaction was markedly increased. Experience with five different lots of D-alanine from four commercial sources indicated that substantial and varying negative bias (up to -10%) could be introduced into the blank-corrected ALT activity, depending on the lot of oalanine used. Although the IFCC procedure for ALT mentions the possibility of this L-alanine contamination, we believe that the degree of contamination in commercial reagents is underestimated. Analyzing the five lots of D-alanirle for Lalanine, we found the magnitude of negative bias to be correlated directly with L-alanine contamination. Here, we describe a quick, sensitive assay based on coupled reactions of L-amino acid oxidase/peroxidase for quantifying L-alanine in the concentration range of 0-15 mmol/L without a sampledilution step. Results by this alternative L-alanine assay agreed well with those recommended in the IFCC ALT procedure. Further examination suggested an even simpler solution to the L-alanine contamination problem, because we found no difference in the blank-corrected ALT activity determined in Tris HCI buffer, with or without D-alanine (free of Lalanine). We therefore propose that D-alanine be omitted from the IFCC reference ALT procedure. AddItIonal Keyphrases: variation, source of
-
coupled enzymat-
ic reaction
-
-
-
of L-
CLINICALCHEMISTRY, Vol. 35, No. 1, 1989 153
L-AAO
alanine in n-alanine. Finally, we evaluated the implication of omitting r)-alanine from the specimen blank reactions (C and D). Materials and Methods For the IFCC ALT assay, we used either the Cary 219 spectrophotometer (Varian Instrument Division, Palo Alto, CA 94303) or the Cobas Bio centrifugal analyzer (Hoffmann-La Roche, Basel, Switzerland). The IFCC method for checking for i,-alanine in n-alanine was performed with the Cary 219 spectrophotometer; for the L-AAOIPOD coupled system we used a Cobas Bio centrifugal analyzer. Reagents. Ti-is; 4-aminoantipyrine (4-A.AP); i-alanine; reduced nicotinaniide adenine dinucleotide (NADH); ALT (EC 2.6.1.2); i.-ainino acid oxidase (EC 1.4.3.2) Type ifi, 1.1 U (phenylalanine at 37 #{176}C)/mg of solid; horseradish peroxidase (EC 1.11.1.7) Type I, 1440 U (at 25 #{176}C)/mg of solid; and pyridoxal phosphate were from Sigma Chemical Co., St. Louis, MO 63178. Lactate dehydrogenase and 2-oxoglutarate were from Boehringer Mannheim, Mannheim, F.R.G. The 2-hydroxy-3,5-dichlorobenzene sulfomc acid (HDBS) was from Research Organics Inc., Cleveland, OH 44125. Two different lots of n-alanine were from Sigma Chemical Co., and one lot each was obtained from Aldrich Chemical Co., Milwaukee, WI 53233; Eastman Kodak Co., Rochester, NY 14650; and ICN Biomedicals Inc., Costa Mesa, CA 92626. Dade “Moni-trol” reference material (XPS 142)used by the American Red Cross as a control for ALT surrogate testings for the non-A, non-B hepatitis-was kindly supplied to us by Dr. Gordon C. Edwards. Procedures. ALT catalytic concentration was measured in selected patients’ samples and the Moni-trol material. All the reagent mixtures were prepared in Tris HC1 buffer according to the IFCC ALT procedure with five different lots of n-alanine from four commercial sources. The Tris HC1 buffer without r-alanine, but containing the other constituents of the reagent mixture, was also included for the estimation of the apparent specimen blank activities in reactions C and D. Absorbance changes at 339 nm at 30.00 ± 0.05 #{176}C were measured with the Cary 219 spectrophotometer. To quantify the i.-alarnne content in the various r)-alanine preparations from four different commercial sources, we used the following reactions: Instruments.
(a) i..-alanine + H20 + 02
(b) H202
S
12
red compound
Figure 1 (left) shows the various amounts of apparent blank activity (C D) for each of the r)-alanine preparations tested with any of three specimens. Thus, for either patients’ or reference sample, we obtained different values (Figure 1, right) for the corrected activity [(A B) (C D)]. This negative bias was as much as 10% or more of the overall activity, depending on the commercial source of the Dalanine. Examining the manufacturers’ stated characteristics of the different lots of n-alanine (Table 1) did not indicate the cause of the bias. Because the ALT is highly specific for tralanine and does not act on n-alanine, we hypothesized that a possible cause of this varying amount of apparent specimen blank activity was contamination of the reagent mixture for reactions C and D with x-alanine. To verify this hypothesis, we checked for and quantified the amount of i..-alanine in n-alanine preparations, using the procedure recommended in the IFCC ALT method and an alternative method based on the L-AAOIPOD system. Both methods gave comparable results for the degree of ialanine contamination of o-alanine -
-
-
-
160 140 S
120 100
10 8
80
6 DFJJ
60
4
40
2
20
0
NH3 + 11202
Results
16
14
4-AAP + HDBS
+
In this L-AAOIPOD system, 5,10, and 15 mmol/L i-alanine standards are used to generate the standard curve. For the reaction, we incubated 200 pL of the reagent [per liter 0.5 mniol of 4-AAP, 1.5 mmol of HDBS, 5.7 kU (40 pg) of peroxidase, 44 U (400 g) of Iamino acid oxidase, and 50 mmol of Tris HC1, pH 7.4] with 80 tL of the D-alanine solution (630 mmol/L) and monitored the change in asorbance at 510 nm. To determine the sensitivity and recovery rate of the assay, we combined various amounts of i.-alanine with the least-contaminated n-alanine (lot B) so that the final concentration of the L isomer ranged from 0.56 to 7.90 mmolJmol of the D isomer. We also determined the i.-alanine content in the various n-alanine lots by the IFCC recommended procedure (1), using various dilutions (up to 200-fold) of the 630 mmol/L Dalanine solution. These dilutions allowed us to determine the maximum concentration of i..-alanine contamination at which the NADH would not be a limiting factor (4).
18 C)
+
pyruvate
DADE STD
PATIENT 1
PATIENT 2
0
DADESTD PATIENT1
SAMPLES
PATIENT2
SAMPLES
Fig. 1. (Left)Variationin specimenblank activities(C D)for three samples(onereferencematerialand two patients’samples)according to source of D-alaninein the reagentmixture,and (right)the blank-corrected ALT activities [(A B) (C D)1determinedwith the IFCC ALT procedure Sources of o-alanine are given inTable 1: Bar A shows actMty in absence of o-alanine(TnsHCI bufferonly) -
-
154
CLINICALCHEMISTRY,Vol. 35, No. 1, 1989
-
-
(Table 1). The results obtained for the quantity of L-alanine in the different n-alanine preparations correspond to the magnitude of the apparent specimen blank activities (C D) as shown in Figure 1 (left). Figure 1 also shows that in all cases we obtained a blank or corrected-activity value for reactions in which r-alanine was excluded similar to that obtained with the least-contaminated r)-alanine, the material from ICN Biomedical Inc. (Table 1). Further to check the reliability of our proposed method,
the L-AAOIPOD
coupled reactions, we supplemented the ICN n-alanine with known amounts of i.-alanine, ranging from 0.56 to 7.90 mmol/mol of r)-alanine and estimated the concentration (Table 2). Analytical recovery ranged from 82% to 112% and averaged 101.8%, with a CV of 10.5%.
Discussion Our experience with these five different lots of n-alanine in the estimation of the apparent specimenblank activities as called for by the IFCC ALT procedure indicatesunacceptable amounts of blank activity in four of the five materials. The effect of over-correction becauseof contamination of the reagent mixture with L-alanine is shown in Figure 1 (right) for two patients’ samples and the Moni-trol material. The implications of this error in value assignment to reference materials or in clinical laboratory service is very apparent. if n-alanine is to be used in this procedure, it should be mandatory that each lot of n-alanine be carefully screened for possible fralanine contamination. In the IFCC ALT procedure the possibility of contamination is indicated, but we believe that the frequency of significant contamination was underestimated by the example given (1). In our investigation only the lot from ICN Biomedicals proved to meet the specifications in the approved IFCC ALT method (Table 1). The present methods, based on specific rotation and thin-layer chromatography, used by the manufacturers for characterizing the r-alanine are inadequate. The chromatographic technique cannot differentiate i- from n-alanine and assessment from specific rotation is too insensitive to detect the degree of contamination we observed. Therefore, we recommend that a more precise method, preferably an enzymatic method based on either the IFCC ALT method or the L-AAOIPOD coupled reaction system, be included in the characterization of i-alanine-free n-alanine. We found that the method recommended in the IFCC procedure for estimation of the fralanine in o-alanine is very sensitive,but somewhat cumbersome, requiring careful adjustment of the enzyme activity concentration, reaction time, and dilution of samples. The L-AAO/POD system
Table 1. Source, Characteristics, and Concentration of L-Alanine in Five Lots of D-Aianine Characterletice
Source iCN (B)
Kodak (C) Aldrich(0) Sigma (E) Sigma (F)
Specific rotation at 15.6#{176}C
-14.2 -14.0 -14.2 -14.5 -14.2
L-Aianine concn,
mmol/moi D-alanine
Purity,
IFCC ALT 99* 98 99* 99+ 99+
0 2.4 ± 0.01 _b
6.0 ± 0.04 5.6 ± 0.03
L-AAO/POD 0 1.9 ± 0.05 5.4 ± 0.05 5.7 ± 0.04 5.6 ± 0.05
IFCC speC. 0.021 As given by manufactureron eitherthe packageinsertor by personal communication witha technical representative. b Quantitynot sufficient.
Table 2. Analytical Recovery of L-Aianine Added to o-Alanine, Measured with the L-AAO/POD Coupled Reaction System L-AIanine, mmoi/moiD-aianins Expected 3.95
Measured 7.44 ± 0.05 4.19 ± 0.06
2.36
2.62
1.59 0.84
1.68 ± 0.06 0.94 ± 0.01 0.46 ± 0.00
7.90
0.56 aMean
±
SD. bfl
=
± 0.07
Recovery, %b 94.2
106.1 111.0
105.7 111.9
82.1
6.
presented here proves to be a suitable independent alternative for enzymatic quantification of the i-alanine. The system is easy to use, automatable, has adequate sensitivity, and is accurate,
as indicated
by the good analytical
recovery for i-alanine-supplemented D-alanine preparations. Although the L-AAO is not specific for j..-alanine, there is no indication in the manufacturers’ characterization by thin-layer chromatography that suggests the presence of other L-amino acids that might serve as substrate for the enzyme. The good agreement between the 1FCC method and the proposed L-AAO/POD procedure shown in Table 1 also that there is no significant contamination of these D-alanine lots by other i-amino acids. The L-AAOIPOD coupled reaction system is thus a valid alternative technique for quickly and accurately checking the r)-alanine for L-alanine contamination. Although the i-alanine contamination can be easily assessed, the high degree of contamination among the commercially available sources raises the question of whether the use of n-alanine in the apparent specimen blank reaction serves any real benefit. In our use of the IFCC ALT method for the unification of results from several different instrument systems (5), we found that omission of n-alamne from the blank reaction led to results equivalent to those in which i.ralanine-free o-alarnne blanks are used. Furthermore, another potential hazard that must be considered when high concentrations of n-alanine are used is the possibility of specimen contamination by n-amino acid oxidase (EC 1.4.3.3). The product of r)-alanine oxidation by this enzyme is pyruvate (6), the same product as that of the transamination reaction involving i-alanine. Although there is no evidence for the presence of n-amino acid oxidase in human serum, quality-control materials might contain significant amounts of this enzyme, which exists in high concentrations in such organs as porcine kidney (6). Finally, elimination of n-alanine from the specimen blank reaction will result in a uniformity among all IFCC enzyme procedures because the isomeric forms of the substrate are not used in the other methods such as the IFCC method for aspartate aminotransferase (7). We therefore recommend that n-alanine be eliminated from the blank reagent in the IFCC ALT procedure. indicates
References 1. Bergmeyer HU, Horder M, Rej R. Approved recommendation (1985) on IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aininotransferass. J Clin Chem Clin Biochem 1986;24:481-95. 2. Braunstein AE. Amino group transfer. In: Boyer PD, ed. The enzymes, Vol. IX, group transfer, Part B. New York: Academic Press, 1973:462-72.
CLINICALCHEMISTRY,Vol. 35, No. 1, 1989 155
3. Barham D, Trinder P. An improved colour reagent for the determination of blood glucose by the oxidase system. Analyst 1972;97:142-5. 4. Bergmeyer HU, Scheibe P, Wahlefeld AW. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin Chem 1978;24:58-73. 5. Okorodudu AO, Valcour An, McComb RB, Bowers GN. The national reference system for alanine aminotransferase (NRS/ALT): evaluation and use of the IFCC reference method for
the numerical unification of ALT results on four analytical instruments usedin daily service [Abstract]. Clin Chem 1988;34:1286. 6. Massey V, Palmer G, Bennett R. The purification and some properties of 1)-amino acid oxidase. Biochim Biophys Acta 1961;48:1-9. 7. Bergmeyer HU, Horder M, Rej R. Approved recommendation (1985) on IFCC method for the measurement of catalytic concentration of enzymes. Part 2. IFCC method for aspartate aminotransfer-
ass.,JClin Chem Clin Biochem 1986;24:497-510.
CLIN. CHEM. 35/1, 156-158 (1989)
Coloscreen VPI Test Kit Evaluated for Detection of Fecal Occult Blood Anthony J. Scriven and Elizabeth M. Tapley
The usefulness of the Coloscreen VP1 test kit in occult blood detection has been examined and compared with our current method. The results show the Coloscreen procedure to be a simple, sensitive method that is much less subject to vegetable peroxidase interference than are other available screening procedures. Additional Keyphrases: colorectal cancer pseudoperoxidase dietaiy restriction screening variation,sourceof The importance of screening for fecal occult blood for early detection of colorectal cancer is well recognized (1). The usefulness of this approach, however, has been hampered by the unreliability of many screening methods (2-4), because the incidence of false-positive results in subjects who are on an unrestricted diet may be very high (5). Most current methods are based on the pseudoperoxidase activity of the hematin portion of the hemoglobin molecule; the oxygen liberated from H2O2 is used to oxidize a dye to a chromogen. Because methods based on this reaction are affected by dietary blood, hemoglobin, and hematin, subjects to be screened should be advised to reduce their intake of redmeat products. Interference from vegetable peroxidase, enzymes present in many types of vegetables and fruit, is also a problem (6, 7). These enzymes demonstrate some resistance to heat denaturation and may not be inactivated by cooking. Dietary restriction of both hemoglobin- and vegetable peroxidaze-containing substances is therefore essential for valid screening for occult blood. Dietary restriction is rarely completely effective, however, because the large number of food products involved generally provokes poor compliance. One approach to reducing interference at the assay stage has been to boil fecal samples thoroughly before analysis, thus inactivating the vegetable enzymes while leaving the pseudoperoxidase activity of hemoglobin largely intact. This procedure is inconvenient when large numbers of samples are assayed; so, also, is a more recently introduced method based on conversion of hematin to porphyrins (8). Test sensitivity is also important. Normal blood loss in feces amounts to 2.5 mL per day (9), whereas >10 mL per
Chemical PathologyLaboratory,Central PathologyLaboratory, North Staffordshire Hospital Centre, Stoke-on-Trent, U.K. Received April 13, 1988; accepted October 11, 1988.
156 CLINICALCHEMISTRY, Vol. 35, No. 1, 1989
day may be considered significant
in the early detection of
colorectal disease. Our current laboratory method, the “Okokit H,” is based on the oxidation of an o-tolidine-like substance, in tablet form, to a green end product. “Coloscreen VPF’ utilizes guaiac (impregnated into test cards) as the pseudoperoxidase substrate and also includes a vegetable peroxidase inhibitor to reduce dietary peroxidase activity. We describe here our evaluation of the Coloscreen VPI test procedure to determine its suitability as a screening method for fecal occult blood. Materials and Methods Specimens Fecal samples (20-50 g) were obtained from routine collections for occult blood testing. These were from hospital inpatients and patients of general practitioners. Before analysis, samples were thoroughly mixed with a wooden spatula. Comparisons between Okokit II and Coloscreen VPI were made with duplicate samples from the same stool specimen. Patients were allowed an unrestricted diet. Fresh samples of blood, collected with EDTA present, were analyzed for hemoglobin (Hb) concentrations with a Coulter Splus (Coulter Instruments, Hialeah, FL 33130), then used as Hb standards. Fecal iron concentrations were determined by the method of Peters et al. (11).
Reagents The Okokit II was obtained from Hughes and Hughes Essex, U.K., and the Coloscreen VPI from Cambridge Self Care Diagnostics Ltd., Ely, Cambridge, Ltd., Romford,
U.K. (manufactured
by Helena Laboratories,
Beaumont,
TX
77704). Vegetable peroxidase was obtained from Sigma Chemicals, Poole, Dorset, U.K. “Analar”-grade water (BDH Chemicals, Poole, Dorset, U.K.) was used throughout. All other reagents were of at least Analar grade. Test Procedures Okokit H is based on the pseudoperoxidase activity of hemoglobin. The liberated 02 is used to oxidize an otolidine-like substance to a green-colored product (the identity of the chromogen is not revealed by the manufacturer). In the test procedure, a small sample of feces, -0.1 g, is smeared onto filter paper provided in the kit, and one drop of reagent A (H202) is added, followed by one drop of reagent B
