NADH system by means of Meldola blue

Clinica Chrmica Actu, 125 (1982) 185- 192 Elsevier Biomedical Press

185

CCA 2289

Sensitisation and visualisation of biochemical measurements using the NAD/NADH system by means of Meldola blue II. Application to the continuous flow determination of plasma glucose and urea J.L. Orsonneau Laboratoire

*, K. Meflah,

de Biochimre Medicale,

(Received

P. Lustenberger,

G. Cornu and S. Bernard

U. E. R. de Medecine et Techniques Mkdicnles, I, rue Gaston Veil, 44035 Names CPdex (France) February

4th, 198 1; revision April 3rd, 198 1) * *

Summary This paper describes the application of the Meldola blue method to the determination in serum of glucose with glucose dehydrogenase and of urea with urease and glutamate dehydrogenase with a continuous flow analyser. It discusses the easy application, reliable results and inexpensiveness of the method.

Introduction As described in Part I [ 11, Meldola blue [2,3] used without tetrazolium salts can be used successfully for the measurement of LDH and CXHBDH activities in serum. This initial finding prompted an extensive search in order to apply the NAD/NADH/Meldola blue system to the enzymatic determination of substrates. We have selected the assay of plasma glucose using glucose dehydrogenase and of plasma urea using urease and glutamate dehydrogenase, these two substances being commonly determined in clinical chemistry. I -

Assay of plasma glucose using glucose dehydrogenase

Materials and methods Materials (the flow diagram is shown in Fig. 1) A sampler set for a cycle of 60 samples per h (sampling/rinsing * To whom correspondence should be addressed. ** The publication of this paper has been regrettably

0009-8981/82/0000-0000/%02.75

delayed

0 1982 Elsevier Biomedical

due to an editorial

Press

error.

ratio

l/l)

177 B 004 diluent

02 PT2

1.00

w wo5te

4t

SamPIe 0.050

-

-

177 0004

02

coplllary tube technicon 0.020 ref 116053605

R 0.160

30’ 0.60

Fig. 1. Manifold

used for the determination

of glucose

in serum.

corresponding to a sample of 25 ~1 is connected to a Technicon P III pump. The pumping and connecting tubes, mixing coils, injectors and other fittings are 2nd generation Technicon components. The reading unit consists of a Corning 254 calorimeter, equipped with a loo-p1 circulation chamber with a built-in de-bubbling device, connected to a Beckman type 600 recorder. The time required between taking of the sample and recording of the result is 8 min 30 s, including 3 min for incubation and 1 min 45 s for the reaction with Meldola blue. Reagents The diluent is composed of distilled water to which is added Triton X-100 (Technicon ref. T21-0188-17) to give a 5% concentration. R,: this represents the Beckman glucose dehydrogenase reagent (ref. 682340): 10 bottles of 15.5 ml. One bottle is enough for at least 90 tests, at a unit cost of about 0.3 F francs. R,: Meldola blue reagent is made up as follows: Meldola blue, Boehringer ref. 258 504, 0.15 mmol; Triton X-100, Technicon ref. T21-0188-17, 25 ml; citrate buffer 0.2 mol/l pH 2.2, qs 1000 ml. This reagent is stable for several weeks at room temperature. Results (a) Determination Repeatability

of precision tests on several

sera

assayed

10 times

in succession

gave

the

187

TABLE

1

RECOVERY

OF GLUCOSE

Serum volume

glucose

Concentrated

(Pl)

Expected serum glucose concentration (mmoI/l)

Serum A 2 2 2 2 2 2 2

0 25 50 100 150 200 250 500

5.43 7.21 8.96 12.31 15.52 18.57 21.49 34.34

1.78 3.53 6.88 10.09 13.14 16.06 28.91

5.43 7.28 9.02 12.37 15.26 18.50 21.27 33.50

1.85 3.59 6.94 9.83 13.07 15.84 28.07

104 102 101 97 99 99 97

Serum 2 2 2 2 2 2 2

0 25 50 loo I50 200 250 500

10.66 12.38 14.06 17.30 20.38 23.33 26.24 38.53

1.72 3.40 6.64 9.72 12.67 15.48 27.87

10.66 12.57 13.93 17.21 10.08 23.22 25.96 37.98

1.91 3.27 6.55 9.42 12.56 15.30 27.32

111 96 99 97 99 99 98

0 25 50 100 150 200 250 500

Il.75 13.45 15.20 f 8.33 21.40 24.32 27.11 39.40

1.70 3.45 6.58 9.65 12.57 15.36 27.65

11.75 13.47 15.20 18.17 21.49 24.33 27.04 38.51

1.72 3.45 6.42 9.74 12.58 15.29 26.76

I 101 100 98 101 100 100 97

solution

(ml)

B

Serum C 2 2 2 2 2 2 2

Glucose added (mmol/i)

Found serum glucose concentration (mmol/i)

Found glucose (mmol/l)

@)

Recovery

following results: for means of 1.89; 5.06, 21.24 and 37.63 mmol, the coefficients of variation were, respectively, 5.26, 1.02, 0.95 and 0.57%. Reproducibility tests carried out on successive days on 25 determinations gave for of variation of 3.07, 2.12, means of 6.40, 9.78, 12.45 and 19.54 mmol,‘coefficients I .96 and 1.81%, respectively. (6) Determination of carry-over Three samples of plasma with glucose tests of 1.7, 4.9 and 10.0 mmol were estimated immediately after a sample with 37.4 mmol; the percentage contamination calculated was, respectively, 1.68, 0.93 and 0.73% (calculated as described by Young and Gochman [4]). Under the same conditions, the percentage conta~nation of a plasma with a level of 20.9 mmol of the first two samples were respectively 1.55 and 1.24%.

Meldola blue met hod

50

30 t

0 20

10

Fig. 2. Comparison between the results and the Meldola blue method.

30

of glucose

40

measurements

50 mm01

obtained

with the GOD-POD

method

(c) Determination of recovery and accuraq The results obtained starting from three sera of different concentrations to which a concentrated glucose solution was added, are given in Table I. The comparison between 290 assays of plasma glucose by continuous flow carried out with the Trinder method [5] as modified (GluciBiotrol ref. A 01538) and with the present method, is shown in Fig. 2, together with the data for the regression line [6].

II -

Enzymatic

assay

of plasma

urea

Materials and methods Materials The manifold used for the assay of plasma urea was identical to that used for glucose, except that the flow rate of the diluent tube is 2.9 ml/mm. The incubation time is reduced to 1 min 20 s, so that the assay takes only 6 min, including 1 min 45 s for the reaction with Meldola blue. Reagents The diluent and reagent R, are the same as those used for glucose. R, is the reagent already described for the enzymatic assay of urea

[7], the

189

concentration of NADH being in this case 0.3 mmol; at least 115 tests can be carried out with one bottle of 20 ml, at a ‘unit cost of less than 0.1 F franc. Results Calibrating standards ranged from 5 to 50 mmol sponded to an increase in absorbance of about 1.0. Determination

urea;

the last point

corre-

of precision

Repeatability tests on several plasmas assayed 10 times in succession gave the following results: for means of 4.00, 9.50, 18.24 and 43.34 mmol, the coefficients of variation were, respectively, 3.33, 1.65, 1.07 and 1.21%. For means of 3.99, 9.46 and 18.59 mmol, reproducibility tests carried out on successive days gave coefficients of variation of 7.23, 2.96 and 2.34%. Determination

Three

TABLE

of carry-over

sera containing

OF UREA

Serum volume

Concentrated urea solution

I 1 1 1 1 1 Serum B 1

1 1 1 1 1 Serum C

1 1 1 1 1

1

18.2 mmol

Expected serum urea concentration (mmol/l)

Urea added (mmol/l)

Found serum urea concentration (mmol/l)

25 50 100 150 200

3.5 5.12 7.48 11.27 18.33 24.78 30.70

0 1.62 3.98 7.77 14.83 21.28 27.20

0 10 25 50 100 150 200

9.60 11.16 13.43 17.08 23.88 30.09 35.78

0 10 25 50 100 150 200

18.10 19.57 21.72 25.17 31.61 37.48 42.86

(al) Serum A

3.8, 9.6 and

were systematically

II

RECOVERY

(ml)

respectively

0 10

Found urea (mmol/l)

(a)

Recovery

3.5 5.15 7.50 11.45 18.45 24.95 30.10

1.65 4.00 7.95 14.95 21.45 26.60

102 100 102 101 101 98

1.56 3.83 7,48 14.28 20.49 26.18

9.60 11.30 13.60 17.20 24.00 30.00 35.60

1.70 4.00 7.60 14.40 20.40 26.00

109 104 102 101 100 99

1.47 3.62 7.07 13.51 19.38 24.76

18.10 19.80 22.10 25.50 31.70 37.20 42.80

1.70 4.00 7.40 13.60 19.10 24.70

116 110 105 101 99 100

190 Meldola method

50

blue

40 -

I

Dam

method

I

I

SMA

40

Fig. 3. Comparison between the results method and the Meldola blue method.

of urea

measurements

tested after a serum containing 42.4 mmol; as before, was 2.34, 1.52 and 1.25%.

6/60

I

50

nmol

obtained

the percentage

with

the diacetyl

contamination,

monoxime

calculated

Determination of recovery and accuracy A concentrated solution of urea was added to three sera; the results given in Table II show satisfactory recovery. Comparison between 202 assays of plasma urea by continuous flow using diacetyl

TABLE

III

INTERFERENCE NH; concentration

BY AM~ONlUM Serum A (mmol)

IONS ON THE UREA Serum B (mmol)

DETERMINATION Serum C (mmol)

(Etmol)

185 367 544 890

3.9 4.0 4.0 4.1 4.4

9.4 9.6

10.0 10.0 10.1

18.8 18.9 19.0 19.2 19.2

191

mono~ne and the other enzymatic with the correlation data.

Meldola

blue method

is shown in Fig. 3, together

Determination of pre-existing ammonium We had to make sure that the test would not be affected by hyperammonemia, which is frequently associated with low values of urea; therefore ammonium ions were added to three sera in increasing proportions; all values were much higher than the physiopathological maximum. The results given in Table III rule out any interference of clinical significance.

Discussion To make sure that there was no interaction with reducing compounds, we used two sera to which were added to one a solution of uric acid and to the other a solution of ascorbic acid. The results shown in Table IV rule out any interference as far as uric acid is concerned; with ascorbic acid the degree of interaction has no clinical significance at normal therapeutic doses. These results were obtained with the glucose manifold with which the dilution of the sample is comparatively unimportant.

TABLE

IV

INTERFERENCE Serum volume (ml)

BY URIC Concentrated ascorbic acid solution (5.7 mmol/l)

AND ASCORBIC

ACID WITH

THE MELDOLA

BLUE METHOD

Serum ascorbic acid concentration (mmolj)

Expected serum glucose concentration (mmol/l)

Found serum glucose concentration (mmol/l)

10.03 9.98 9.93 9.79 9.55 9.12

10.03 9.90 9.90 9.90 9.77 9.37

(al) 1

0

1

5

1

10 25 50 100

0.03 0.06 0.14 0.27 0.52

Concentrated uric acid solution (10 mmoI/l)

Serum uric acid concentration (mmol/l)

Expected serum glucose concentration (mmol/l)

Found serum glucose concentration (mmol/l)

385 620 843 1259 1639 2308

10.37 10.12 9.88 9.43 9.02 8.64

10.37 10.10 9.97 9.57 9.04 8.5 1

1 1 1 Serum volume (ml)

(PI) 1

0

1

25 50

I

1 I 1

loo 150 250

192

These two methods for the assay of plasma glucose and urea are reliable, as shown in particular by correlation analysis with tried techniques such as glucose oxidase and diacetyl monoxine, respectively. Their application is easy, as it requires a simple manifold, without dialysis, the water bath being optional and replaceable by an increase of the incubation time; on the other hand, there is no need for a blank, as even cloudy sera do not affect the assay, owing to the high dilution. Moreover, the two measurements can be associated on a two-channel AutoAnalyzer system, with only three reagents. The inexpensiveness of this technique is noteworthy: the consumption of enzyme reagent is reduced to a minimum, and definitely lower than that of most centrifugal analyzers, in spite of their reputation for saving reagents. This is due to the increased sensitivity brought about by Meldola blue, the price of which is minimal and the consumption of which is very low. This cheapness could be enhanced if shorter cycles were adopted, which is made possible by the very low degree of contamination. These two techniques can be integrated in the programme of a sequential analyzer of the SMA type without any modification. Finally, the use of Meldola blue for the enzymatic determination of other substrates should not present more difficulty than for these two assays. References Orsonneau JL, Meflah K, Lustenberger P, Cornu G, Bernard S. Sensitisation and visualisation of biochemical measurements using the NAD/NADH system by means of Meldola blue. I. Principle and application to the continuous flow measurement of LDH and aHBDH activities in serum. Clin Chim Acta 1982; 125: 177-184. German Patent: Indikator zur Bestimmung der reduzierten Pyridin &enzyme. Boehringer Mannheim GmbH; 1959: 410. Bergmeyer HU. Methods of enzymatic analysis. Verlag Chemie, Academic Press, NY, 1974; 1: 136-144. Young DS, Go&man N. Standard methods of clinical chemistry. Academic Press, NY, 1972; 7: 303-304. Trinder P. Determination of glucose in blood using GOD with an alternative oxygen acceptor. Ann Clin Biochem 1969; 6: 24-27. Westgard JO, Hunt MR. Use and interpretation of common statistical tests in method comparison studies. Clin Chem 1973; 19: 49-57. Orsonneau JL, Meflah K, Cornu G, Bernard S. Utilisation dun reactif unique pour ie dosage enzymatique de I’uree sanguine et urinaire. Ann Biol Clin 1980; 38: I39- 142.