Rapid automated enzymatic measurement of phenylalanine in plasma and blood spots

ELSEVIER

Clinica Chimica Acta 218 (1993) 207-214

Short communication

Rapid automated enzymatic measurement of phenylalanine in plasma and blood spots Richard P. Taylor*, Ian C. Smith, Susan J. Standing Dept. of Clinical Biochemistry, John Radcliffe Hospital, Headingeon, Oxford, OX3 9DU, UK (Received 20 May 1992; revision received 22 February 1993; accepted 18th March 1993)

Key words: Phenylketonuria; Dietary monitoring; dehydrogenase; Autoanalysis; Chemistry; Clinical

L-Phenylalanine; Phenylalanine

1. Introduction

The measurement of blood phenylalanine is an essential tool in determining the appropriate phenylalanine intake for phenylketonuric patients. Recent work has advocat~ the maintenance of blood phenylalanine concentration in early childhood in the range 120-300 ~mol/l [I]. Phenylalanine can be measured in serum or plasma or in dried blood spots collected on filter paper. The techniques in common use are fluorimetry [2], HPLC with [3] or without derivatisation [4,5], or automated amino acid analysis [6]. Fluorimetry can be automated [7] but is susceptible to interference, especially for blood spots [8,9]. Both HPLC and amino acid analysis are accurate and precise but require expensive equipment, special expertise and are often lengthy procedures. None of these methods offers the combination of rapid, accurate and technically straightforward analysis. Recently phenylalanine dehydrogenase (L-phenylalanine: NAD + oxidoreductase (deaminating) EC 1.4.1.-) has been used in assays generating NADH from the NAD+-dependent oxidative deamination of phenylalanine [10], and others employ the additional step of the reduction of a tetrazolium salt to a formazan dye catalysed by diaphorase [!1,12]. Phenylalanine dehydrogenase activity linked to a tetrazolium/intermediate electron accepter detector system has been produced in kit form * Corresponding author. 0009-8981/93/$06.00 tc~ 1993 Elsevier Science Publishers B V. All rights reserved. SSD1 0009-8981(93)05572-V

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as the Quantase TM phenylalanine assay (Porton Cambridge Ltd., Newmarket, UK) for dietary monitoring using plasma or blood spots in manual and microtitre plate formats [13]. We have assessed the manual formulation and developed a rapid and technically simple automated protocol using low sample and reagent volumes on a centrifugalanalyser. As the manual assay appears to overestimate serum phenylalanine compared with HPLC [I 3,14], our assessment included comparison with HPLC and necessitated the development of modified protocols. 2. Material aad methods

All reagents with the exception of the Quantase phenylalanine kits were purchased from Sigma Chemical Co. Ltd., Poole, UK, or BDH Ltd., Poole, UK. The papers (HMR 101/6) for blood spot collection were of the type used for neonatal screening and were obtained from HMSO Forms Centre, Oldham, UK.

2.1. HPLC Plasma and blood spot phenylalanine were measured by HPLC as described elsewhere, with water (50/d) added to the plasma or blood spots before addition of the protein precipitant [I 5].

2.2. QuantaserM phenylalanine dehydrogenase Reagents. The components are (i) lyophilised L-phenylalanine dehydrogenase, (ii) buffered enzyme diluent, (iii) buffered !yophilised NAD +, (iv) colour reagent containing buffered tetrazolium salt, intermediate electron acceptor and detergent and (v and vi) 200 and 1,000/~mol/i phenylalanine calibrators. Automation of original manual protocol Each vial of enzyme was reconstituted with 0.7 ml of enzyme diluent and each coenzyme vial with 2.2 ml of distilled water. For plasma assays enzyme and coenzyme reagents were mixed in the ratio 1:4, respectively, and for blood spots in the ratio 1:3. The manufacturer's proposed manual protocol was adapted for the Cobas Fara (Roche Products Ltd, Welwyn Garden City, UK) using these combined reagents as shown in Table |(a). Absorbance measurements were made at 570 nm, at 25°C. End-point calculations were based on the difference in absorbanc¢ between auxiliary reading M I and absorbance reading 13. In separate experiments the period of colour development was varied from 90 to 240 s. Modified protocols for the Fara. Enzyme and coenzyme reagents were re,~onstituted as described but were not combined before analysis. The coe~zyme was designated as Parallel Pipetted Reagent, enzyme as Start Reagent ! and colour reagent as Start Reagent 2. Instrument settings for the modified assays a,e given in Table l(b). For the blanked plasma assay three versions of the method with virtually identical parameters were entered into the analyser memory and designated 'Precalibration', 'Test' and 'Blank' (Table l(b)), Before analysing patient's samples the Precalibration method was run with 1,000/~mol/I standard to obtain a calibration factor relating absorbance to phenylalanine concentration. This was entered into the Test and Blank versions of the method parameters and samples were then analyscA. Enzyma-

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209

Table I Cobas Fara settings for original and modified phenylalanine assays Parameter Calibration mode (i) Precalibration assay (ii) Test analysis (iii) Blank analysis Reagent blank Analysis pa Sample volume (#1) Reagent volume (pl) (i) enzymc/coenzyme

SR2

(ii) coenzyme only Temp delay (s) Incubation (s) Auxiliary reading MI (s) Start reagent ! (td) (i) colour reagent (ii) enzyme reagent for Test or enzyme diluent for Blank Incubation (s) Auxiliary reading MI (s) Start (colour) reagent 2 0 d )

!

I n c u b a t i o n (s)

A

First reading (s) Number of readings Interval (s)

T I SRi

1

Calibration Standard ! (~mol/I, duplicates) Factor

(a) Original assay

(b) Modified assays

-Slope average Reagent/diluent

Slope average Factor Factor Reagent/diluent

PS 10/BS 40 b

PS 10/BS 40

I00 -5 PS 300/BS 600 PS 295/BS 595"

--

PS 80/BS 75 5 5 --

90 --

-PS 20/BS 25

120 --

PS 300/BS 600 PS 295/BS 595 90 120 0.5 13 10

--

---

0.5 13 10

1,000¢ d

1,000c d

Calculation [] Absorbancc reading 13-Absorbancc reading M! up, Parallel (i.e. simultaneous) pil~tting of sample and reagent. bps, plama assays; BS, blood spot assays. CCalibrant value entered in PCA-precipitated plasma and blood spot assays. For blanked plasma assay calibrant value is entered for Pr~alibration assay only. dFor Test (enzyme) and Blank assays in blanked plasma assay only; set to factor obtained from Precalibration assay.

tic (Test) and Blank assays were performed concurrently in the same rotor withm the same run for each sample to minimise imprecision. In the Blank assay the start reagent (SRI) comprised enzyme diluent without enzyme, as shown in Table l(b). The instrument was set to calculate automatically the difference between the Test and Blank assays for each sample. For plasma assay after protc'-~ precipitation, plasma (100 ~,1) was mixed with 100 tA of 0.59 tool/! perchloric acid (PCA), vortex mixed for 10 s, left to stand for 15 rain and centrifuged at 8,000 × g for 5 rain. The clear supernatant was assayed for phe-

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nylalanine by the modified protocol in Table l(b) without a blank assay. Each run was calibrated using the standard (l,000/~mol/I) diluted I:1 with 0.59 mol/I PCA. For the assay of blood spots discs of 6 mm diameter were punched and eluted in 200 pl of 0.184 mol/l Analar trichloroacetic acid (TCA). The tubes were vortex mixed for 10 s and the eluant shaken to the bottom of the tube to ensure that the spots were fully immersed. The tubes were left for 30 min, re-mixed and centrifuged if necessary. The eluates were analysed without a Blank run by the modified protocol with a sample volume in the assay of 40 pl. Each assay was calibrated with the 1,000 pmol/I standard diluted 1:30 with 0.184 mol/l TCA.

2.3. Smnples analysed Venoas or fingerprick capillary blood samples were obtained from normal individuals and phenylketonuric patients on dietary therapy. For recovery experiments 5 retool/! phenylalanine in water was added to whole blood or plasma. Patient's or control plasma samples were stored at -20°C and blood spots at room temperature until analysis.

2.4. Statistical procedures Correlation data were analysed by Deniing's double-error regression [16].' 3. Results and discussion

3.1. HPLC The HPLC method had excellent precision and recovery as reported previously [15]. An aqueous sample with a phenylalanine concentration of 2,000 ;tmol/I de~ermined by gas chromatography mass spectrometry gave a HPLC result of 1,1998 ~mol/I,

+i

3.2. Phenylalanine dehydrogena~+e Original plasma protocol. The original assay used combined enzyme/coenzyme reagent which has a reported stability of approx. I day at 2-8°C. In the modified assays the reagents were combined only in the reaction cuvette, thereby pr01on~ing the life of the reconstituted reagents to at [east 3 weeks at 4°C [14], an advantage if analysing small batches. The assay +~howed ~ood linearity between 63 and 2,000 #mol/l using aqueous phenylalanine solutions. The regression equation was: measured phenylalanine = (0.962 x theoretical phenylalanine) + 18.2/smol/I; r = 1.000. Within- and between=batch imprecision were excellent (Table 2). There was no significant interference from bJlirubin or tyrosine, confirming other reports [12,14]. Phenyltactate did not interfere at concentrations up to 50 ~mol,'l, the range observed in phe~ylketor, una [17], nor did lactate up to i0 mmol/l. A comparison of the pheny|a!anine dehydrogenase method according to the manufacturer's proposed protocol with HPLC for patient's plasma samples showed

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Table 2

Within- and between-batch imprecision for plasma and blood spot assays Within-batch

Mean

Between-batch

S.D.

(~mol/l) Original plasma protocol a

Blanked plasma protocol b

PCA-precipitated plasma b

TCA-extracted blood spots c

115 459 917 35 248 560 122 339 770 122d 271 1081

2.30 2.75 18.3 !.54 5.70 21.8 3.68 7.04 20.4 14.5 25.3 45.4

C.V.

Mean

(%)

(pmol/I)

2.0 0.6 2.0 4.4 :.3 3.9 3.0 2.1 2.7 il.9 9.4 4.2

112 469 898 47 186 627 106 332 736 40.4 286 1089d

S.D.

C.V.

~%) 4.48 9.38 2~9 11.3 20.5 43.9 11.7 11.3 21.7 9.36 25.1 57.6

4.0 2.0 3.0 24.0 11.0 7.0 11.1 3.4 2.9 23.2 8.8 5.3

aWithin-batch n = 8, between-batch n = 4. bWithin-batch and between-batch n = 10. %Vithin.batch n = 9, between-batch n = I 1 except d n -~ 12.

an excellent correlation between the methods (r = 0.992) with a slope of 0.954, but there was a positive intercept of 130/tmol/I. The latter is identical to that observed in a detailed repoit on the manual method [14] and was suggested to be a consequence of using the tetrazolium reaction. In addition there was continuing ¢olour development beyond the recommended 120 s at a rate equivalent to 4.5 ttmol/l of phenylalanine per minute of fut'ther reaction time (Table 3). This effect was independent of the phenylalanine concentration. Patient's and bovine control specimens behaved similarly, but it was not observed in aqueous phenylalanine solutions. Modified protocols. In view of the positive intercept and the observed continuing rise in absorbance with plasma samples, modified protocols were developed. In the modified protocols the continuing colour development beyond 120 s was eliminated.

Table 3 Effect of changing the timing of the final absorbance reading on the phenylalanine result in a plasma sample with a phenylalanine conc~ntration of 115 ~mol/I (n = 9)

Time interval to final absorbanee reading (s)

90 120 150

Phenylalanine result as a percentage of result at 120 s

Time interval to final absorbanee reading (s)

Phenylalanine result as a percentage of result at 120 s

98.0 100.0 102.5

180 210 240

104,5 107,0 108.9

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212

The blanked plasma assay gave excellent agreement with HPLC in the recommended monitoring range, with the intercept reduced to 22 #mol/i, an acceptable level (Fig. la). The within-batch imprecision was excellent, with a C.V. of less than 5% across the concentration range. The between-batch C.V. was higher than in the original protocol, rising above 11% at concentrations below 186 ttmol/I (Table 2), but it is likely that the impre~.ision could be reduced by increasing the sample volume above the 10 ttl used, to increase the colour yield. The calculation factor from the precalibration assay relating absorbance to phenylalanine concentration was 1,487 (C.V. = 0.62%) over 11 assays, showing excellent calibration stability and indicating that it would not be necessary to recalibrate for every batch. The blank assay can run concurrently on the Fara analyser and the results can be calculated automatically, but these features are not essential. The precalibration run is necessary to derive a calculation factor, as a calibrant cannot be used in the blank assay without enzyme. The blanked method has the advantage of simplicity and no additional manipulative steps. A serum-based calibrant has been suggested as an alternative to individual specimen blanks [14], but this would not correct for variability in the non-specific reaction between specimens. Protein precipitation with PCA followed by neutralisation of the supernatant with potassium carbonate or bicarbonate before assay has been used successfully to minimise the non-specific reaction~ of the enzyme [1 I, 12]. In initial experiments we found that omitting the neutralisation step did not affect the results, but that its inclusion increased the imprecision of the assay. For plasma assayed without neutralisation after protein precipitation the PCA supernatants gave an excellent correlation with HPLC with an intercept of 29 ~mol/I (Fig. lb). The slope of the regression line ~vith HPLC was 1.006, so there was a positive bias of approximately

g 1200rm~ o

i°4,.

0 ~ o

2

o

Plasma pha (HPLC) (ixmol/I.)

o

o

Plasma phe (HPLC) (pmoI/L)

'~ Blood apot phil (HPLC) (Ixmol/ll.)

Fig. I. Correlation of Porton Quantase enzymatic protocols (y) with HPLC (x) for human plasma or blood spots, a: O, blanked plasma; .v = 0.935x + 21.9; r = 0.994; n = 77; 95% confidence interval (C.i.) of slope = 0.91 I-0.958; 95% C.]. o£ intercept = 9.31-.~4.4. b: A, PCA-precipitatod p]asma; y = 1.006x + 28.6; r = 0.995; n = 30; 95% C.[. of slope = 0.969-1.04; 95% C.]. of intercept -- 0.927-56.2. ¢: D, blood spot~; .~'= l.G97x - 21.4; r = 0.983; n = 54; 95% C.I. of slope = 1.042-I.L55; 95% C.I.. of intercept = -50.5-7.81.

R.P. Taylor et al, / Clin. Cliim. Ac~a 218 (1993) 207-214

213

30/zmol/l at all measured phenylalanine concentrations relative to HPLC, but this is unlikely to impair the utility of the assay. Both within- and between-batch imprecision were good (Table 2), being only slightly inferior to those of the original protocol. The within-batch C.V. was 3% or less. The between-batch C.V. was 11.0% at 107/zmol/l but fell to 3.4% at 332/zmoi/i. The recovery of 221/zmol/l of added phenylalanine was 100.8% (S.D. = 3.93%, n = 19) and for 645 /zmol/i was 99.2% (S.D. = 3.39%, n = 19). The calculation factor (not used in these experiments) was 2,925 (C.V. = 0.85%) over ten assays. Protein precipitation introduces an additional step but it is rapid and simple, removes most of the interference and results in a more robust assay with excellent precision. Phenylalanine in blood spots extracted with TCA as recommended by the marmfacturer but assayed by the automated modified Fara method was in good agreement within the recommended monitoring range with paired blood spots extracted with PCA and assayed by HPLC (Fig. lc). Good agreement has also been reported for the manual assay [13]. Blood spot within- and between-batch imprecision were similar (Table 2), and acceptable for phenylalanine monitoring. The between-batch imprecision was better than that of the blanked plasma assay but worse than for the PCA.precipitated plasma assay. This can be explained by the smaller quantity of sample analysed in the cuvette, which was only 20% of the amount in the original plasma assay, together with the additional error associated with collection, punching and elution of blood spots. The recovery of 250 ~mol/I of added phenylalanine was 101.2% (S.D. = 7.18%, n = 16) and for 1,000 tzmol/I was 106.3% (S.D. = 4.70%, n = 17). The calculation factor (not used in these experiments) was "/,918 (C.V. = 5.07%) over 17 assays. Twice weekly phenylalanine monitoring has been advocated Ill, and most laboratories have few specimens in each batch. The m~iomated enzymatic assay is a straightforward procedure ideal for such a work pattern. The reagents are stable and easy to prepare. The reagent cost is approximately £2 per test which is appreciably more than for HPLC, but this must be set against the significant saving in staff time and the greater costs of maintaining and operating HPLC. Local circumstances will influence the cost/benefit relationship. The modified plasma and blood spot methods developed on the Cobas Fara show excellent agreement with a HPLC method, and ~haveacceptable imprecision and accuracy. They use small sample volumes, are technically straightforward and could readily be adapted to many other currently available automated analysers. The manufacturer's recommended protocols have been modified in the light of this work [18]. The blanked plasma assay is faster and easier to perform than the PCAprecipitated plasma assay but the latter has lower between-batch imprecision. They are all rapid techniques suitable for the routine monitoring of dietary therapy in phenylketonuria according to recently reco~mended criteria. 4. Acknowledgements

We thank Porton Cambridge Ltd. for provision of the reagents and Dr. M. McLaren of Roche Products Ltd. and Dr. J. Land of this department for their advice.

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5. References I Smith I, lkasley MG, Ades AE. Intelligence and quality of dietary treatment in phenylketonuria. A~'ch Dis Child 1990;65:472-478. 2 McCamanMW, Robins E. Fluorimetric method for the determination of phenylalanine in serum. J Lab Ciin Med 1962;53 ~85-890. 3 Rudy JL, Rutledge JC, Lewis SL. Phenylalanine and tyrosine in serum and eluates from dried blood spots as determined by ~e+,rsed-phase liquid chromatography. Clin Chem 1987;33:1152-1154. 4 Hilton MA. Liquid-c'~romatographic direct determination of phenylalanine and tyrosine in serum or plasma, with a~fication to patients with phenylketonuria. Clin Chem 1982;28:1215-1218. 5 Atherton ND, Ct~n A. HPLC measurement of phenylalanine in plasma. Clin Chem 1988;34: 2241-2244. 6 lknson JV, C.,rmick J, Patterson JA. Accelerated chromatography of amino acids associated with phenylketor~t~ia, leueinosis (maple syrup urine disease), and other inborn errors of metabolism. Anal Bioc~em 1967;18:481-492. 7 Blau K. ++:~termination of phenylalanine in filter paper blood spots by a simplified automated fluorimt~,.+icmethod without dialysis. Clin Chim Acta 1983;129:!97-200. 8 Am[,~x~eJA. Report on a cooperative study of various fluorometric procedures and the Guthrie b~+~rial inhibition assay in the determination of hyperpheny[f.laninemia. Health Lab Sci t97.~,i0:180-187. Gerasimova NS, Steklova IV, Tuuminen T, Fluorometric method for phenylalanine microplate assay adapted for phenylketonuria screening. Clin Chem 1989;35:2112-2115. I0 Hummel W, Schtitte H, Kula MR. Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase. Anal Biochem 1988;170:397-401. I I WendelU, Hummel W, Langenbeck U. Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase. Anal Biochem 1989;180:91-94, 12 WendelU, Koppelkamm M, Hummel W. Enzymatic phenylalanine estimation for the management of patients with phenylketonuria, Clin Chim Acts 1991;201:95-98. 13 CampbellRS, Hollifield RD, Varsani HJ et al. Development of an enzymatic colorimetric assay for phcnylalanine. Poster C74, In: Martin SM, Halloran SP, eds. Proceedings of the ACB National Meeting, Glasgow, 13-17 May 1991, London: R~M 1991~117. 14 Campbell RS, Brearley GM, Varsani H ct al, ikvclopmcnt and validation of a robust specific enP,yme mediated assay for phenylalanine in serum, Clin Chim Acts 1992;210:197-210. 15 StandingSJ, Taylor RP, Phenylalanine: application of a simple HPI.C technique to its measurement in dried blood spots, Ann Clin Biochem 1992;29:668-670+ 16 Strike PW, Medical laboratory statistics. Bristol: J Wright & Sons, 1981:173-189. 17 Clemens PC, Schlinemann MH, Hoffmann GF, Kohlschiitter A, Plasma concentrations of phenyllactic acid in phcnyiketonuria. J inhe~'it Metab Dis 1990;13:227-228. 18 QuantaseTM phenylalanine product information, Newmarket, UK: Porton Cambridge Lid,, 1992.