Note on Slot's method for the specific determination of creatinine

CLINICA

CHIMICA

ACTA

NOTE

ON SLOT’S

493

METHOD

FOR

THE

SPECIFIC

DETERMINATION

OF

CREATININE

11. GIIXFNETTEII,

Z. JANW?O\‘.I\

AND 1. &KVINIiOVi

Institute for Cavdiovasculav Research, Prague (Czechoslovakia) (Received

April

10th. 1967)

SUMMARY

Slot’s method for true creatinine determination has been improved to give quantitative recoveries of creatinine and reproducible pseudocreatinine values. The present modification meets analytical criteria, it is simple and specific and suitable for either manual or automated serial true creatinine determinations.

Measurements of creatinine are usually based on the Jaffe reaction’ utilising the ability of picrate to form an orange colour with creatinine. Unfortunately, the reaction lacks specifity. To obtain true creatinine values one should eliminate the interference of the so-called pseudocreatinine* (nonspecific chromogen). This can be done through absorption of creatinine on Lloyd’s reagent2-@, or by other meansloF1l. Methods of this kind introduce a more complicated handling of the samples which considerably decreases their efficiency in routine use. Recently, Slot has published a simple method for the specific determination of creatinine in serum12. He found that only the true creatinine colour equivalent disappears after acidification of the sample. In this way the apparent creatinine value could be corrected for the nonspecific chromogen by two simple spectrophotometric measurements, i.e. in the alkalineI and acidified sample. When testing the same samples by the Slot method in parallel with the well proved Brod and Sirota technique3, the former method gave satisfactory results only over the normal range of serum creatinine concentration, while much lower values were seen at high creatinine concentrations. In this paper, the reasons for the low yield at elevated creatinine concentrations are discussed and a modification of the Slot technique is presented which satisfies analytical requirements. METHOD

Proposed

experimental

modification

of the Slot procedure

(based on results

presented

in the

part)

* Pseudocreatinine has the same meaning terms total creatinine, apparent creatinine

as the nonspecific or total chromogen

chromogen fraction. are interchangeable. Clin.

Similarly,

the

Chim. Acta, 17 (1967) 4g3-498

494

GKAFNETTEK

et al.

Reagents: 1.2% picric acid in water: some batches of pircric acid give high reaction blanks. In that case a different batch, or picric acid recrystallized from glacial acetic acid should be used. 0.75 N sodium hydroxide. 5% sodium tungstate. Picrate reagent: I vol. of 1,296 p icric acid is mixed with I vol. of 0.75 N NaOH just before use. 0.66 N sulphuric acid. Standard creatinine solutions : 2.5 ml of a stock (IOO rngyb) creatinine solution are made up to IOO ml with 0.05 N sulphuric acid. This 2.5 rngyb solution is further diluted with 0.05 N sulphuric acid to the following concentrations: 1.5; 1.25; 1.0; 0.75; 0.5 and 0.25 mg%. If 3 ml of each diluted solution are run in the same way as samples, their spectrophotometric readings correspond (with respect to the sample dilution in tungstate filtrates) to 6, 5, 4, 3, 2 and I rng% of creatinine in serum or plasma.

Preparation

of plasma 0~ serum tungstate jiltrate vol. of plasma or serum is diluted with I vol. of water and I vol. of 5% sodium tungstate. Then drop by drop and under continuous mixing, I vol. of 0.66 N sulphuric acid is added. About IO min later, the mixture is filtered (using, e.g. Schleicher and Schuell Filter Plisses). I

Calibration cwve 2 ml of picrate reagent are added to 3 ml of each standard creatinine solution. Colour is read at 500 rnp after 45 min against a blank containing 3 ml of 0.05 N sulphuric acid and 2 ml of picrate reagent. With our spectrophotometer (SPEKOL) and using r-cm cuvettes, the curve was linear up to 6 rng% of creatinine in plasma.

Creatinine content in serum OYplasma

For each sample, 2 tubes (A and B) containing 3 ml of the tungstate filtrate are prepared. Two ml of the picrate reagent are added to the set of tubes A and the developed colour is read after 45 min at 500 rnp against a blank containing 3 ml of 0.05 N sulphuric acid and 2 ml of picrate reagent (total chromogen readings). With a suitable time delay-so that timing of measurements would not interfere-2 ml of picrate reagent are also added to the set of tubes B, including a new blank. Instead of reading the colour, 0.1 ml of glacial acetic acid is added to all tubes B after 45 min as well as to the new blank. The true creatinine colour equivalent disappears. The remaining optical density (nonspecific chromogen) is read at 500 rnp 5 min after the addition of acetic acid against the acidified blank. The difference between the readings of total and nonspecific chromogen corresponds to the true creatinine, the concentration of which is obtained using the calibration curve. All analyses are preferably performed in duplicate. Creatinine content in wine

Urine is diluted with water 100-250 times according to the urine density. Three ml of the diluted sample are mixed with 2 ml of the picrate reagent. Colour density is

CREATININE

DETERMINATION OF SLOT

495

read as in the case of serum samples (total chromogen). Nonspecific chromogen is not evaluated since its concentration is negligible in urine with respect to the high content of creatinine. EXPERIMENTAL

Table

The original procedure of Slot did not yield sufficient recovery (see example in I) especially at high creatinine concentrations. This was mainly due to a low

TABLE

1

CREATININERECOVERIES

AS

OBTAINED

BY

THE

OF

SLOT

Total found mg96

Recovery SO

q.00

2.52

2.16

2.26

4’ 5’

.~~~._ I

Sample creatinine content, mgo,b

Creatinine a.dded 2mgOa I

o.go

2

1.15

Serum

samfile

METHOD

content of sodium hydroxide in the final reaction mixture, since, when 4 ml of tungstate filtrate are mixed with I ml of picrate reagent the resulting pH is below optimum. Colour development proceeds slowly for more than go min. Tegger-Nilsson stated that pH 12.4 was optimal and we, as well as otherslo were able to confirm this. Hence, substitution of 0.75 N by z N sodium hydroxide in the Slot procedure corrected both the reaction course and the recoveries. However, the same could be achieved when only 3 ml of the tungstate filtrate were mixed with z ml of picrate reagent (I.z~/~ picric acid and 0.75 N sodium hydroxide, I: I). Since the latter ratio has been commonly used in many of the hitherto developed methods, we preferred to include it into our working procedure as described in METHODS. Naturally, at this ratio addition of more acid than proposed by Slot will be needed in the step for evaluation of the nonspecific chromogen fractions. For this reason the reaction mixture should be brought to approximately pH 4.0. Even small differences in the amount of sulphuric acid added could lead to considerable variations in the final pH. This would cause difficulties since, in our experience, a pH below 4.0 resulted in turbidity. Therefore we tried to substitute 0.25 ml of I N sulphuric acid by a smaller amount of glacial acetic acid. A well chosen excess of acetic acid was expected to buffer the mixture in combination with the simultaneously formed sodium acetate. In fact, when different amounts of glacial acetic acid were added to reaction blanks or samples (Table II), only minor pH changes occurred with increasing T.L\BLE II RELATION

OF

THE

NONSPECIFIC

CHROMOGEN

READING

TO

pH

Five 3 ml aliquots of a serum tungstate filtrate were mixed with 2 ml of picrate reagent and colouration left to proceed for 45 min. Then the stated amounts of glacial acetic acid were added and optical densities read 5 min later. -__ Addition of glacial pH Optical density after acetic acid (ml) 5 min at 500 mf.i ualuf ____~ 0.05 0.033 4.55 0.10 0.032 4.15 0.15 0.03 I 3.95 0.20 0.035 3.85 0.25

0.041

3.58

Clin. Chim. Acta, 17

(1967)

493-498

GRAFNETTER

496 TABLE

et

al.

111

REPRODUCIBILITY

OF

pH LOWERING

BY

GLACIAL

ACETIC

ACID

Total chromogen colours were developed in IO samples (originating from different sera) as described in Table Il. Then, 0.1 ml of glacial acetic acid was added and the pH measured.

SLWlpk

PH

I

4.22

2

Santple

4

4.18 4.15 4.15

5

4.16

3

pH

6

4.19 4.20

; 9

4.18 4.18

IO

4.1.5

volume of acetic acid. Additions of 0.1 ml of glacial acetic in our procedure, in a pH near to 4.2 (Table III). human

IV). When different

T.ABLE

CREATININE

BROD

SIROTA

AND

between

the results

obtained

human sera were analysed

by the two methods

with our procedure

for total

IV

APPARENT

Pvesent

resulted,

We compared the levels of the total chromogen (apparent creatinine) in 60 sera as obtained by our procedure and by the Brod and Sirota3 technique,

and found very good correlation (Table

acid constantly

LEVELS

AS OBTAINED

BY

THE

PRESENT

_ Method of Brod 6 Sirota mg 7’0

method

Method of Rrod CC Sivota

mg9

WY%

Present method mg “6

0.75 0.84 0.88

0.88 0.68 0.76

I.34 I.34 1.36

0.90 0.93 0.97

0.72 0.80 0.84

I.38 I.39 I.41

0.98

1.02

I.42

1.00 I.02 1.07 1.07 1.08

0.92 0.88 1.04 0.96 0.96

1.43 I.43 1.48 1.48 1.51

1.10 I.12 I.12 1.12 I.13 1.15

1.00 0.96 1.08 1.04 1.08 1.08

I.54 I.59 1.62 I.64 1.67 I .68

I.44 I.32 1.60 1.64 1.36 1.56

1.18 1.21 I.22

1.04 1.20 1.12

I.68 I.76 I.87

1.23 I.24

I.52 1.08

I .93 2.26

1.16 I.20 I.32 1.36

1.28 1.32 1.40 I.32 1.20 I.36 1.48 I.48

I.!24

1.16

2.39

1.60 1.64 I .92 2.00 2.24 2.52

I.25 1.26 I.27

I.12 1.08 1.20

2.38 2.48 2.64

2.44 2.52 2.64

I.32 I.33 I.34

1.28 I.24 I.20

3.54 5.15 5.89

3.52 5.64 5.68

Clin. Chim. Acta, 17 (1967) 493-468

METHOD

AND

BY

THE

METHOD

OF

CREATININE TABLE

DETERMINATION

497

V

RECOVERIES

OF

Serum sample .4 B B C D E F F G G H I Ii

and

OF SLOT

CREATININE

AS

TESTED

1.28

4.07 0.96

5.31 3.65 6.22

4.97 1.56 1.56 0.96

5.64 2.23 2.23 I.42

3.63 5.95 2.81

I.‘#.?

2.85

1.16

1.41 2.85 1.41

4.39 2.63 4.08 2.58

1.18

nonspecific

chromogen,

levels

within

amounted

to

31%)

between

showed

high

TABLE

of

often

the

two

seen

99 99 IO1

99 96 IO2

97 98 99 101

I”4 100 99

constituted

4.4-40.8~/~

0.75-7.10

mg%.

the

chromogen.

total

in diabetics;

but

Almost

of the former

all nonspecific Exceptionally

on the whole,

at total

chromogen high

there

was

values

no direct

parameters.

experiments precision

latter

the range

IO-Z~~/~

were

Recovery

the

Recovery %

3.71 4.59 6.04

I.44 2.87 1.41

I.23

relation

PROCEDURE

Total found m&Y %

Z.IO

(e.g. 41%,

PRESENT

Creatinine added, mg %

2.16 3.00

values

THE

Sample creatinine content, mg%

L.IO

chromogen

BY

(Table

of the

above

V)

as well

proposed

as reproducibility

studies

(Table

VI)

procedure.

VI

REPRODUCIBILITY CHROMOGEN

OF THE TOTAL AND NONSPECIFIC

DETERMINATION

IN

ONE

SERUM

Total chvomogen ma?;

Nonspecific chromogen mgO,b

1.770 1,780 1.710

0.210

I.759 1.720

0.260 0.210 0.230 0.240

1.780 1.720 I ,690 1.700 1.710

0.210 0.230 0.210 0.210 0.210

Mean : i_ SD.:

Mean: J- S.D.: _.

I.733 0.034

SAMPLE

0.222 0.017

DISCUSSION

Slot’s nine

reportI

determinations

to fade

after

opened

the possibility

utilising

acidification.

the

However,

if the reaction

conditions

could

nine.

done

by

This

was

here

property

of the

this ingenious

be modified

changing

of a simple

to yield

the ratio

but specific

method

creatinine-picrate suggestion

was

quantitative

between

colour

tungstate

only

for creaticomplex

of real

recoveries filtrate,

value

of creatisaturated

Clin. Chim. Acta, 17 (1967) 49x-498

GRAFNETTER

498

et id.

picric acid and 0.75 IL’ sodium hydroxide from 8: I : I (Slot) to the hitherto well proved 3: I : I ratio, giving proper alkalinity for colour development. Under these conditions, however, acidification had to be rechecked to guarantee full disappearance of the true creatinine colour equivalent. We have replaced 0.25 ml of I N sulphuric acid by 0.1 ml of glacial acetic acid which has the advantage of also improving the buffering of the final pH. Results obtained by the herein presented modi~cation correlated very well with those obtained on the same material by the method of Brod and Sirota (Table IV). However, the Brod and Sirota method gave slightly lower values in a majority of cases. This may be explained by the fact that in our procedure standard samples and blanks were prepared in 0.05 N sulphuric acid in order to match the acidity of tungstate filtrates. Without matching in the Brod and Sirota method, the slightly higher alkalinity in the standard samples may have been of significance in the final evaluation of creatinine concentrations. It was observed that aqueous creatinine solutions developed full colour within 15-20 min, but colour developed differently in tungstate filtrates so that the maximum was reached only after 40 min. Therefore, 45 min was chosen in our procedure as the best time for optical density readings. Our experience concerning pseudocreatinine concentrations agrees well with previous reports based on different methods3s4*6T13. We believe that our modification of Slot’s method, giving quantitative performance over a wide range of creatinine concentrations, is simple and specific, suitable equally well for manual and for automated creatinine determinations. REFERENCES I M. JAFFE, 2. Ph.ysysiol.Chem., IO (1886) 391. 2 H. BORSOOK, J. Biol. Chem., 110 (1935) 481. 3 4 5 6 7 8

J. J. J. 0.

R. 2.

g Ii. TO E. II A. 12 C. 13 H.

BROD AND J. H. SIROTA, J. C&n. Invest., 27 (1948) 645. BROD, in XlinickQ Fysiologie a Pathojysiologie Ledvin, Statni zdr. nakl. Praha, T. CLARKE, Clin. Chum., 7 (19Gr) 371. D. DOOLAN, E. L. ALPEN AND G. B. THEIL, Am. 1. Med., 32 (rg6z) 6.5. S. HARE, Pvoc. Sot. Exptl. Bid. Med., 74 (rgso} i48. MILLER ANU B. F. MILLER, PYOC. Sot. Ex$tE. Biol. Med., 78 (1951) 471. W. BONINESS AND H. TAUSSKY, J. Biol. Chem., 158 (1945) 581. POLAR AND T. METCORF, C&n. Chem., II (196.5) 763. C. TEGGER-I?ILSSON, Stand. J. C&z. Lab. invest:, 13 (1961) 326. SLOT, Stand. J. CEin. Lab. Invest., 17 (1965) 381. N. HAUGEN AND E. M. BLEGEN, Stand. J. C6in. Lab. Invest., 5 (1953) 67.

C&z. Chim. Acta, 17 (1967) 493-498

1962.