Serum alanine assay with an enzymic micromethod (L-alanine dehydrogenase)

95

Clinica Chimica Acta, 0 ElsevierlNorth-Holland

86 (1978)

95-100

Biomedical

Press

CCA 9402

SERUM ALANINE ASSAY WITH AN ENZYMIC MICROMETHOD ( L-ALANINE DEHYDROGENASE)

A. BERG, W. GEROK Xfedizinischen (G.F.R.)

(Received

Klinik

December

and J. KEUL der Universitiit

Freiburg,

Hugstetter

Strasse 55, 7800 Freiburg

i. Br.

23rd, 1977)

Summary L-Alanine was measured by an enzymic micromethod in serum after treatment with perchloric acid, as well as after ultrafiltration through collodium membrane filters. Serum L-alanine was also determined by an automated column chromatographic. technique. Using the enzymic method, spuriously increased L-alanine levels could be obtained due to the absorbent reaction between hydrazine and NAD-containing reagents. It could be shown that this absorbent reaction depends on the pH value of the test solution. In contrast to supernatant diluted by perchloric acid, ultrafiltrates gave L-alanine data equivalent to those by column chromatography, since conditions of reaction time and pH value could be kept identical for blank, standard and ultrafiltrate solutions.

Introductions The amino acid alanine has been demonstrated to be a central substrate between protein catabolism and carbohydrate generation in man. Alanine is released by muscle tissue, taken up by the liver and directly converted to glucose. Many laboratories have obtained rates of gluconeogenesis from alanine in relation to other substrates and hormones in normal and diseased subjects [l61. Concerning this, it is also evident that amino acids, and in particular alanine, are not unimportant in starvation, diabetes and pregnancy as well as during prolonged and exhaustive physical exercise. The study of L-alanine levels in blood and of its metabolism in various physiological and pathological situations had to be carried out with the time-wasting procedure of column chromatography determinations. The enzymic alanine assay described by Williamson [ 71 was inaugurated primarily for hepatic tissue and pure alanine solutions. The work of other teams (ref. 8; Poortmans, J., personal communication) and

our own experience seem to show that this enzymic assay employed for serum determination produces data different from those obtained by column chromato~aphy. Therefore it was the purpose of the foltowing study to describe the enzymic micromethod for serum alanine determination and the conditions under which data are reproducible and in good agreement with those of column chromatography. Materials and methods In 10 healthy male adults (20-27 years) blood was drawn after overnight fasting at 9 a.m. from the cubital vein: avoiding stasis and without anticoagulant. Serum was separated by centrifugation and stored until analysis in small plastic tubes at -35°C. For determination, serum was deproteinised by pipetting in 1 volume of perchloric acid (6%) and centrifuged for 5 min (12 000 rev./min); in all cases serum was also deproteinised by ultrafiltration through collodium membrane filters (collodium bags SM 13200; Fa. Sartorius, Gottingen) (30 min, 3500 rev./min). L-Alanine was measured in the supernatant, the ultrafiltrate or standard solutions using the following recipe:

-

ml

Hz0 (dilution) Addition Hydrazine-Tris buffer, pEI 10 NAD

Standard

Supernatant

Ultrafiltrate

Blank

1.000 0.100

0.900 0.200

1.000 0.100

1.100 __

1.000 0.100

1.000 0.100

1.000 0.100

1.000 0.100

_

_..

-

The concentrations of the solutions used were made up as described by Williamson [7] for the alanine enzymic assay. The initial absorbance at 334 nm was determined (Eppendorf mercury-lamp photometer) and the interval of time between pipetting NAD-solution and the first reading was kept exactly constant for all samples. The dehydrogenating reaction was started by addition of L-alanine dehydrogenase (0.005 ml, 150 U/ml; Fa. Boehringer, ~~annheim, No. 102636). The reaction took place at room temperature and was generally finished within 60 min. The calculation of L-alanine resulted from the difference of absorbance after extrapolation to zero time using 6.15 (dimension, 1 mol-l cm-’ X 106) as NADH extinction coefficient [9]. For the pH-dependent reactions the pH value was calibrated with KOH (2 M) and adjusted using a digital pH-meter (Fa. Knick, Berlin; type-No. 640) with a microeleetrode (Fa. Ingold, Frankfurt; type-No. 405-MS). Serum L-alanine was additionally determined in the ultrafiltrate by the automated ion-exchange chromatographic analyser Technicon TSM. Mean values, standard deviation and percentage of variation were calculated; correIations were computed as linear regression functions. Results Under the reaction solutions (0.10~1.000

conditions described previously L-alanine mmol/l) was found to 100 per cent:

of standard

TABLE

I

RECOVERY Initial

OF

ALANINE

ADDED

Alanine

serum

TO

SERUM

added

akline

to serum

(mmol/l)

(mmol/l)

Total

Recovery

alanine

(rnrnOl/l)

(S)

95.0

0.364

0.200

0.554

0.352

0.300

0.648

98.7

0.333

0.400

0.734

100.3

0.384

0.500

0.872

97.6

0.476

0.600

1.070

99.0

0.461

0.800

I.249

98.5

0.443

1.000

1.435

99.4

x-i

TABLE

98.4

SD.

f_ 1.7

II

VARIABILITY

OF

SERUM-ALANINE

Number

ENZYME

of

Serum

ASSAY Variation

alanine

determinations

(mmol/l)

H.W.

5

0.376

i 0.011

B.A.

5

0.396

+ 0.006

1.5

E.B.

5

0.374

t 0.009

2.4

R.L.

5

0.326

t 0.008

2.5

A .W .

5

0.337

_t 0.008

2.4

u

AA

(%I 2.9

2.3

t S.D.

t 0.5

b 4

;

0.1 -

9.2

10.0 0.0% ,

1

60

0 Fig. of 9.2

1. Change and

10.0.

of absorbance (a) addition

AA/t of

(min)

NAD,

(b)

in blank addition

t(min) solution of

containing

Galanine

hydrazine

dehydrogenase.

and

NAD-reagent

at a PH

98

m mol/I ADH

l

0.1

/

mmol/l

/

/’

0.5oc

/

,*’

r-o.992

.3oc

I 0.1

APH

Fig. 2. Increased measuring of L-&nine (Y: mmol/l) blank and test solution: Y = (0.311X + 0.006) mmol/l Fig. 3. Correlation between serum L-alanine enzymic assay (Y; L-alanine dehydrogenase 95% confidence interval (dotted line).

0.500

,300 as a function L-z&nine.

of PH difference

(S:

APH) between

levels measured by column chromatography (S: col.) and the ADH): Y = (1.0035 + 0.016) mmol/l L-&nine. within the

L-alanine mmol/l measured = (1.00 standard added - 0.001) mmol/l L-alanine; r = 0.999; N = 10. The recovery of L-alanine added to serum was 98.4 per cent as demonstrated in Table I. The variability of the serum-alanine enzymic assay is shown in Table II. We found that there was a pH-dependent reaction between hydrazine and NAD (Fig. 1); if pH values of blank, standard and serum reaction solutions are different, no reproducible and exact results can be expected. Fig. 2 shows that this error is correlated to the pH differences between the reaction solutions investigated. Using serum ultrafiltrates, deproteinising with the perchloric acid was not necessary and pH values of the different reactions are essentially the same. In this case the data from column chromatography and those from the enzymic procedure are in very good agreement (Fig. 3). The mean value for the serum alanine in 10 adults amounted to 0.427 * 0.091 mmol/l by the enzymic micromethod as compared to 0.410 + 0.090 mmol/l by column chromatography; the variation between the two methods was 4.1 per cent. In contrast to this, an increased concentration of 0.558 + 0.074 mmol/l alanine was measured in the supernatants of the sera being treated with perchloric acid. The pH difference between standard, blank and ultrafiltrate (pH about 9.80) and supernatant final solution (pH about 9.35) amounts in general to approximately 0.45. When the increased values were corrected with regard to the pH-dependent error (Fig. 2), the mean values resulted in the same quantity (0.419 + 0.074 mmol/l) as that calculated for the ultrafiltrates.

99

Discussion From other NADH-dependent reactions it is well known that solutions containing hydrazine and NAD form an optically absorbing compound; this absorbance is still important at 334, 340 or 365 nm [lo]. In general the unspecific blank reaction starts very rapidly for the first minutes and is subsequently retarded to a reaction rate of 0.010 to 0.040 AA/60 min. Therefore it is absolutely necessary that the time interval after NAD-addition be kept constant for blank and test reactions. This is relevant in all continuous assays started with NAD and hydrazine solutions. Inaccurate time-measuring leads to an irreproducible or incorrect determination of the substrate quantities. In the case of the alanine enzymic assay it seems that the reaction rate of absorbance is dependent on the pH of the actual final solutions. In the first minutes after adding NAD the absorbing reaction proceeds much more rapidly at pH 10.0 than at pH 9.2 (Fig. 1). The reaction rate is further influenced by the addition of L-alanine dehydrogenase. Whereas after pipetting alanine dehydrogenase the rate of blank-absorbance AA is not altered at pH 9.2, it is reduced up to pH 9.6 and finally reversed at pH 10.0; usually within 20 minutes, the absorbance of the blank (final pH 9.8) returns to the zero-time value. In contrast, the absorbing reaction persists in the test containing sera treated by perchloric acid. For this reason it is important that reactions of blank, standard and sera are carried out under two conditions: (1) within constant time intervals; (2) at equivalent pH values. Since exact neutrahsation turns out to be laborious, determination of L-alanine in serum ultrafiltrates appears to be practical and satisfies the conditions demanded above. A further advantage is that the deaminating reaction of L-alanine dehydrogenase takes place at near optimum pH level [ 111. Using the ultrafiltrates the data from the enzymic assay are in very good agreement to those

TABLE

III

SERUM

OR

PLASMA

TOGRAPHY)

BY

ALANINE

VARIOUS

VALUES

IN

HUMAN

SUBJECTS

AT

AUTHORS Alanine

n

(mmol/l)

Ref.

w

S.D.

Arterial Carlsten

et al.

4

0.322

0.075

car1sten

et al.

3

0.256

0.051

19

0.255

0.009

11

0.236

0.023

Felig

et al.

Wahren

et al.

12 13 *

14 15

VeWXls Bergstrijm Brodan Katz

et al.

London Poortmans wurtman * S.E.M.

et al.

et al. et al. et al. et ai.

21

0.330

0.098

16

8

0.366

0.061

17

12

0.352

0.059

18

6

0.262

n.i.

19

12

0.442

0.062

20

23

0.460

0.100

21

REST

(COLUMN

CHROMA-

100

from column chromato~aphy; further, they compare well with the results published by various authors for human venous sera or plasma (Table III): a mean value (N = 82) of 0.385 ? 0.096 mmol/l [16-211. In contrast to the venous levels arterial blood contains significant less L-alanine (p < 0.001): mean value (N= 37) 0.257 of-0.039 mmol/l [ 12-151, accordingly a difference of 33.2 per cent. This difference in concentraiion is due Lo the known muscular release and hepatic uptake of this amino acid [2,14,19]. When changes in serum or plasma alanine are to be investigated, it is necessary to take blood under comparable conditions, since physical exercise as well as the individual nutritive status influence the alanine balance. If blood is prepared immediately after sampling, identical concentrations of r.,-alanine are found in serum and plasma P81. Acknowledgement The authors are much indebted formed the column chromatographic

to Mrs. R. Kajewski analyses.

for having kindly

per-

References 1

Fefig,

Ph.,

2

Fe&g,

Ph. (1973)

3

Fernandes,

4

Chiasson,

5

Vidnes.

6 7

Kim,

Y.J.,

J. and J.L.,

Garber,

A.J., Invest.

Williamson, 1727,

58,

D.H.

Schutgens,

R.&H.,

Bergmeyer,

H.U.

11

1747-1753,

Bergmeyer. meyer,

12

Cat&ten.

ed.).

A.,

Lab.

23,

J. Clin.

Invest.

51,

1195-1202

in Methoden

der

1149-1156

B.C.

Invest. J.V..

CT..

36,

and

Lacy,

W.W.

(1975)

Diabetes

24,

5’74-584

347-356

Haymond,

M.W..

enzymatischen

K.

and

456-457.

Ha&en.

B.;

and

Klin.

(1974)

Chemie,

Gawehn.

F.A.

Chem.

H.U.

Verlag pp.

Beemer,

Z. Klin.

Bergmeyer,

H.U.,

H.U.,

Invest. 13

and

Metabolism

Santiago,

Awie,

E.

pp.

(1972)

Pagliara,

A.S.

and

Kiwis.

D.M.

(1976)

Analyse

(Bergmeyer,

H.U.,

ed.),

(1977)

Chim.

pp.

1724-

Weinheim

(1975)

Berndt,

R.

Sinclair-Smith,

J. Clin.

(1974)

ed.).

(1974) J.E.,

Hendler,

179-207

7-15

Chemie.

9

and

22,

Ph.E.,

Cryer.

Verlag

V.

W.

&and.

8 10

Blom,

biljenquist,

J. (1976)

J. Clin.

Lynch,

Metabolism

Berntssen,

Biochem.

W.J.M.

13,

in Methoden

der

Clin.

Acta

80,

l-5

507-508 enzymatischen

Analyse

(Bergmeyer,

H.U.,

Weinheim Grassl,

Verlag

M.

(1974)

Chemie,

Jagenburg,

in Methoden

der

enzymatischen

Analyse

(Berg-

Weinheim

R.,

Svanborg,

B.,

Jagenburg,

A.

and

WerkG,

L.

(1962)

&and.

J. Clin.

Lab.

(1965)

Aeta

14,185-191

Carlsten,

A.,

Physic&

&and.

14

Fe&,

15

Wahren,

16

BergstrGm,

17

Brodan,

18

Katz,

Htiggendat,

Ph. and Wabren, J., V., N.R.

19

London,

20

Poortmans,

21

Wurtman,

Fe&, J.,

Hallgren.

J. (1971)

Ph.,

Fiixst,

Kuhn.

Hendler, P.. Noree,

E.,

and Keck,

D.R., J.R., R.J.,

J.,

R.,

Svanborg,

A.

and

WerkG,

L.

64.434-447

Foley, Siest, Rose,

Pechar, M.

(1977)

T.H. G.,

J. Clin. R. and L.O.

50.2703-2714

and

G. (1973)

Vinnars,

J. and Tomkov&, J. Klin.

and Webb, Galteau.

C.M.,Chou,

Invest. Ahiborg,

C.G.

M.M. C. and

D.

Chem.

(1976)

Klin.

(1965) and

Biochem.

F.F.

0.

Physiol.

J. Appl.

34, 36,

Physiol.

838-845 693-697 35,

69-77

15.89-91

208,

588-589

(1974)

(1968)

Physiol.

J. Appl. Eur.

Nature

Houot,

Larin,

J. Appl.

E. (1974)

New

Eur.

J. Awl.

Engl.

J. Med.

Physiol. 279,

32,

143-147

171-175