- Email: [email protected]
Application of heyrovsky's inulin method to automatic analysis
CLINICA CHIMICA
ACTA
APPLICATION
OF HEYROVSKY’S
f-53
INULIN
METHOD
TO AUTOMATIC
ANALYSIS
J. K. DAWBORN*
Medical
Unit, University College Hospital, London (Great Britain)
(Received
August
21st, 1964)
SUMMARY
Heyrovsky’s method for the estimation of inulin has been adapted for automatic analysis using a Technicon Autoanalyser **. Hydrolysis of inulin to fructose before the separation of protein by dialysis enables the estimation to be made directly on plasma. Reproducible results were obtained on plasma samples with satisfactory recovery of added inulin.
INTRODUCTION
In 1956 Heyrovskyl described a method for the determination of inulin in urine and protein-free filtrates of plasma. Inulin was hydrolysed to fructose by the addition of concentrated hydrochloric acid and a purple colour developed by incubation with 3-indolylacetic acid. Heyrovsky found the colour reaction sensitive, interference by glucose was small and temperature control less critical than in other published methods of determination. Concentrated hydrochloric acid is essential for efficient colour development and performed the additional function of hydrolysis in the original method. In the automated method to be described, protein is separated by dialysis. As the membrane used is impermeable to inulin, the inulin must be hydrolysed to fructose before dialysis. The time of dialysis is constant and the dialysate therefore contains a constant proportion of fructose which is estimated calorimetrically. MATERIAL
AND METHODS
Reagents
(i) Concentrated hydrochloric acid (A.R.); (ii) Hydrochloric acid (A.R.), 0.5 N containing 0.5 ml “Brij-35”** per litre as a wetting agent; (iii) 3-indolylacetic acid, 0.5 g/100 ml in absolute ethanol. * British Council Commonwealth Scholar. * * Technicon Instruments Company Ltd.
Clin. Chim. Acta,
12
(1965) 63-66
A~lalytical fwocedurr Tlic analyses were* l)erformcd on a Technicon cuit illustrated in Fig. 1 which also includes details containing
inulin
were diluted
with 0.5 A’ hydrochloric
Autoanal\3cr of flow rates.
ubing thcs flex\- c,ir.The plasma s;arnl)l(:s
acid and the inulin hydrolysed
to fructose by heating at ho” for IO min in the first coil of a double-coil heating bath*. Protein was then removed by dialysis across a “cuprophane” membrane* at 37“ and colour developed by heating chloric acid and 3-indolylacetic bath.
The intensity
RECORDER
the protein-free dialvsate with concentrated hrdroacid at Go” for 8 min in the second coil of the heating
of colour is estimated
photometrically
at ,?zo nip and rccordetl.
@ TJgon pulw rupprermr
@ cllarspllt,ruppmror
CaaJnlum
W
Fig. I. Flow diagram including flow rates. Hydrolysis and protein separation is carried out in circuit X using the first coil of the heating bath. The second coil of the heating bath is used for colour de\~clopmcnt. RESULTS
ANI) DISCUSSIOiX
Most experiments were conducted using r-ml samples and a sampling rate of zo/h. At this rate there was excellent reproducibility and complete separation between sample peaks over the entire range (o-1.0 mg/ml). At a sampling rate of 40/h separation between samples was less complete but quite adequate if successive samples differed by 0.3 mg/ml or less. Seven standards were used to cover the range from o-1.0 mg/ml inulin. The calibration curve was linear for the range o-o.5 mg/ml and above this deviated only slightly from linearity (Fig. 2). Recovery exfwiments Measured aliquots of heat alkali stable pooled human plasma and the recovery of levels of o.oog-0.015 mg/ml were small when of 0.3-0.5 mg/ml. The experiment covered a * Technicon
Instruments
Clin. Chinz. Ada,
12 (1965)
Company 63-66
Ltd
inulin * were added to lo-ml samples of added inulin estimated. Plasma blank compared with expected plasma levels range of plasma inulin levels from O.I-
AUTOMATED
INULIN
65
METHOD
0.75 mg~ml. The mean recovery at a sampling rate of zo/h was g8.2% (96.7-100.8~~) and at 40/h, 99.3% (g6.4-roz%) (Table I). In three different experiments a total of 33 single estimations of the recovery of various amounts of inulin were made at a sampling rate of 20/h and 15 estimations at a sampling rate of 40/h. The mean recovery of added inulin at 20/h was g&5”/ f SD 2.27 and at 40/h was 99.2% & SD 1.84.
Fig. 2. Calibration TABLE
curve
which
is linear
over the range
o-o.5
mg/ml
inulin.
I
RECOVEXY OF INUL~N
ADDED
TO
IO
ml 0F
PLASMA
AT
SAMPLING
RATES
0F
20
AND
40
s.weLEs
PERHOUR
Inulin added
mg
Calculated
recovery
Giamples/h -~, mg inulin
I
2.5 3.5 5.0 5.0 7.5
Hydrolysis
%
0.972 0.97 2.52
100.8
2.52
100.8
3.42 3.42 4.83 4% 4.94 4.85 7.41 7.32
97.7 97.7 96.7 97.7 99.0 97.7 98.7 97.0
97.2 97.0
__...._ 40 samples/h --m,g inulin 0.99 I .02 2.42 2.52 3.37 3.37
5.01
4.90 5.01 5.01 7.60 7.65
% 99 I02
96.7 100.7
96.4 96.4 100.2 98.0 100.2 100.2 101.2 102.0
and dialysis
The cuprophane membrane used for dialysis is not permeable to inulin and virtually no colour developed if the acid hydrolysis was omitted. Hydrolysis of inulin to fructose appeared to be almost complete using 0.5 IV hydrochloric acid at 60” for IO min. Neither increasing the strength of the acid to 1.5 N nor doubling the incubation time increased the final colour. Increasing the temperature from 60” to 95” increased the final colour by 5-1oO/~,but the higher temperature caused coagulation of protein which blocked the dialyser. The intensity of colour at 60” was about 20% C&z. Chim.
Acta,
IZ (1965)
63-66
of that developed when dialysis was omitted, so that approximately fructose crossed the membrane during the lo-min period of dialJ4l;.
zo’),, ot tins
The conditions for final colour development in the procedure adopted seem to be optimal. Incubation for longer times or at higher temperatures increased the total cnlour, but this was entirely offset by an equal rise in the blank \Auc. :\s noted b!. Heyrovsky, colour decreased if the final concentration of h~droc~hloric acid fell below 3o;j;X w/v.
It was not possible to incory:orate digestion with sodium hydroxide into the method because sodium chloride precipitated in the presence of concentrated hydrochloric acid. The presence of sodium chloride crystals in the flow cell interfered with light detection by the photocell of the calorimeter. This made the method unsuitable for the estimation of inulin in tissue extracts where destruction of labile chromogens by alkali is essential. The anthrone colour reaction”, although less sensitive, is more satisfactory in this respect. ACKNOWLEl?CEMEKTS
critical
I wish to thank discussion.
REFEKENCES
the British
Council for financial
support
and Dr. E. J. Ross for
ACTA
APPLICATION
OF HEYROVSKY’S
f-53
INULIN
METHOD
TO AUTOMATIC
ANALYSIS
J. K. DAWBORN*
Medical
Unit, University College Hospital, London (Great Britain)
(Received
August
21st, 1964)
SUMMARY
Heyrovsky’s method for the estimation of inulin has been adapted for automatic analysis using a Technicon Autoanalyser **. Hydrolysis of inulin to fructose before the separation of protein by dialysis enables the estimation to be made directly on plasma. Reproducible results were obtained on plasma samples with satisfactory recovery of added inulin.
INTRODUCTION
In 1956 Heyrovskyl described a method for the determination of inulin in urine and protein-free filtrates of plasma. Inulin was hydrolysed to fructose by the addition of concentrated hydrochloric acid and a purple colour developed by incubation with 3-indolylacetic acid. Heyrovsky found the colour reaction sensitive, interference by glucose was small and temperature control less critical than in other published methods of determination. Concentrated hydrochloric acid is essential for efficient colour development and performed the additional function of hydrolysis in the original method. In the automated method to be described, protein is separated by dialysis. As the membrane used is impermeable to inulin, the inulin must be hydrolysed to fructose before dialysis. The time of dialysis is constant and the dialysate therefore contains a constant proportion of fructose which is estimated calorimetrically. MATERIAL
AND METHODS
Reagents
(i) Concentrated hydrochloric acid (A.R.); (ii) Hydrochloric acid (A.R.), 0.5 N containing 0.5 ml “Brij-35”** per litre as a wetting agent; (iii) 3-indolylacetic acid, 0.5 g/100 ml in absolute ethanol. * British Council Commonwealth Scholar. * * Technicon Instruments Company Ltd.
Clin. Chim. Acta,
12
(1965) 63-66
A~lalytical fwocedurr Tlic analyses were* l)erformcd on a Technicon cuit illustrated in Fig. 1 which also includes details containing
inulin
were diluted
with 0.5 A’ hydrochloric
Autoanal\3cr of flow rates.
ubing thcs flex\- c,ir.The plasma s;arnl)l(:s
acid and the inulin hydrolysed
to fructose by heating at ho” for IO min in the first coil of a double-coil heating bath*. Protein was then removed by dialysis across a “cuprophane” membrane* at 37“ and colour developed by heating chloric acid and 3-indolylacetic bath.
The intensity
RECORDER
the protein-free dialvsate with concentrated hrdroacid at Go” for 8 min in the second coil of the heating
of colour is estimated
photometrically
at ,?zo nip and rccordetl.
@ TJgon pulw rupprermr
@ cllarspllt,ruppmror
CaaJnlum
W
Fig. I. Flow diagram including flow rates. Hydrolysis and protein separation is carried out in circuit X using the first coil of the heating bath. The second coil of the heating bath is used for colour de\~clopmcnt. RESULTS
ANI) DISCUSSIOiX
Most experiments were conducted using r-ml samples and a sampling rate of zo/h. At this rate there was excellent reproducibility and complete separation between sample peaks over the entire range (o-1.0 mg/ml). At a sampling rate of 40/h separation between samples was less complete but quite adequate if successive samples differed by 0.3 mg/ml or less. Seven standards were used to cover the range from o-1.0 mg/ml inulin. The calibration curve was linear for the range o-o.5 mg/ml and above this deviated only slightly from linearity (Fig. 2). Recovery exfwiments Measured aliquots of heat alkali stable pooled human plasma and the recovery of levels of o.oog-0.015 mg/ml were small when of 0.3-0.5 mg/ml. The experiment covered a * Technicon
Instruments
Clin. Chinz. Ada,
12 (1965)
Company 63-66
Ltd
inulin * were added to lo-ml samples of added inulin estimated. Plasma blank compared with expected plasma levels range of plasma inulin levels from O.I-
AUTOMATED
INULIN
65
METHOD
0.75 mg~ml. The mean recovery at a sampling rate of zo/h was g8.2% (96.7-100.8~~) and at 40/h, 99.3% (g6.4-roz%) (Table I). In three different experiments a total of 33 single estimations of the recovery of various amounts of inulin were made at a sampling rate of 20/h and 15 estimations at a sampling rate of 40/h. The mean recovery of added inulin at 20/h was g&5”/ f SD 2.27 and at 40/h was 99.2% & SD 1.84.
Fig. 2. Calibration TABLE
curve
which
is linear
over the range
o-o.5
mg/ml
inulin.
I
RECOVEXY OF INUL~N
ADDED
TO
IO
ml 0F
PLASMA
AT
SAMPLING
RATES
0F
20
AND
40
s.weLEs
PERHOUR
Inulin added
mg
Calculated
recovery
Giamples/h -~, mg inulin
I
2.5 3.5 5.0 5.0 7.5
Hydrolysis
%
0.972 0.97 2.52
100.8
2.52
100.8
3.42 3.42 4.83 4% 4.94 4.85 7.41 7.32
97.7 97.7 96.7 97.7 99.0 97.7 98.7 97.0
97.2 97.0
__...._ 40 samples/h --m,g inulin 0.99 I .02 2.42 2.52 3.37 3.37
5.01
4.90 5.01 5.01 7.60 7.65
% 99 I02
96.7 100.7
96.4 96.4 100.2 98.0 100.2 100.2 101.2 102.0
and dialysis
The cuprophane membrane used for dialysis is not permeable to inulin and virtually no colour developed if the acid hydrolysis was omitted. Hydrolysis of inulin to fructose appeared to be almost complete using 0.5 IV hydrochloric acid at 60” for IO min. Neither increasing the strength of the acid to 1.5 N nor doubling the incubation time increased the final colour. Increasing the temperature from 60” to 95” increased the final colour by 5-1oO/~,but the higher temperature caused coagulation of protein which blocked the dialyser. The intensity of colour at 60” was about 20% C&z. Chim.
Acta,
IZ (1965)
63-66
of that developed when dialysis was omitted, so that approximately fructose crossed the membrane during the lo-min period of dialJ4l;.
zo’),, ot tins
The conditions for final colour development in the procedure adopted seem to be optimal. Incubation for longer times or at higher temperatures increased the total cnlour, but this was entirely offset by an equal rise in the blank \Auc. :\s noted b!. Heyrovsky, colour decreased if the final concentration of h~droc~hloric acid fell below 3o;j;X w/v.
It was not possible to incory:orate digestion with sodium hydroxide into the method because sodium chloride precipitated in the presence of concentrated hydrochloric acid. The presence of sodium chloride crystals in the flow cell interfered with light detection by the photocell of the calorimeter. This made the method unsuitable for the estimation of inulin in tissue extracts where destruction of labile chromogens by alkali is essential. The anthrone colour reaction”, although less sensitive, is more satisfactory in this respect. ACKNOWLEl?CEMEKTS
critical
I wish to thank discussion.
REFEKENCES
the British
Council for financial
support
and Dr. E. J. Ross for