- Email: [email protected]
A rapid photometric micromethod for serum lipase determination
CLINICAL CHIMICA ACTA
CCA
199
4761
A RAPID
PHOTOMETRIC
MICROMETHOD
FOR
SERUM
LIPASE
DETERMINATION
U. LIPPI,
G. STEVI’INATO
Depavtmrnt (Received
0-f Pathology, Septcmbcr
I,
AND G. GliIDI
P. Cosma Hospdal,
35012 Camposampiero
(Italy)
1971)
A new micromethod for the determination of serum lipase activity is presented. Small quantities of serum are incubated for 30 min at 40’ in a substrate consisting of an olive oil suspension in the presence of deoxycholic acid. The pH is 8.5. The hydrolyzed fatty acids are determined with a photometric technique, with a slight modification of the method of Duncombe. The normal values are lower than 20 I.U.
INTRODCCTION
Several methods have been published for the determination of serum lipase, but none is fully satisfactory. Several suggested modifications of the Cherry-Crandalll method increase the sensitivity and reduce the time of incubation. Pancreatic lipase is able to hydrolyse esters of long chain fatty acids containing 8 to 18 carbon atoms. Therefore as substrate for lipase, esters of long chain fatty acids are to be preferred, but even triolein (olive oil) is hydrolyzed to a small extent by esterases. According
to Sarda
and Desnuelle2,
lipase
acts exclusively
on insoluble
sub-
strate. Fatty acids released by the hydrolysis of triglycerides by lipase are measured 6s7 techniques or by turbidimetrys. Several by titrimetic1T3p5 and spectrophotometric photometric methods are accurate and sensitive; however the use of very delicate and unstable substrates, an overlong incubation time and the sophisticated manipulations required to perform a lipase analysis preclude their use in many laboratories. A new photometric micromethod for serum lipase estimation is presented in this report. PRINCIPLE
Serum lipase hydrolyses olive oil triglycerides. One of the products of reaction are free fatty acids which are measured calorimetrically according to the method of Clin. Chim. Acta, 37 (1972) 199-~02
LIPPI
200
ct a/.
Duncombeg, with slight modifications. The substrate composition and the pH, the serum-substrate ratio, the incubation time and the effect of temperature have been evaluated by Henry’“, by Massion and Seligson7 and by several other workers”-I:$. Our present method is able to reduce the volume of serum (0.10 ml), and substrate (1.0 ml). Incubation time has been reduced to 30 min. The method is ver\r sensitive, accurate and it gives reproducible available from manufacturers+.
results. The reagents
are easil!, prepared
or in part
REAGESTS
(I) Substrate: Purified olive oil, 5 ml is homogenized (at o‘ in a I’otter~Elvelljcnl glass homogenizer fitted with a Teflon pestle) in IOO ml of a solution containing 0.242 g”/o of Tris-(hydroxymethyl)-aminomethane, 0.350 g:“,; of deoxycholic acid and of 0.02 go/, of ascorbic acid. The pH is adjusted to 8.50 with I X HCl or I ~‘v:?r’aOH. The emulsion can be used for 20~30 days if stored at +-lo. Olive oil is completely freed from fatty acids by passing it through alumina (I?. Merck, AC Darmstadt, So. (2) Chlorofon~, reagent grade. (PH , ‘fi:) Cofili,6~ reagmt: 0.27 Rl copper nitrate in 0.45 31 triethanolamine in butanol. (4) 9. o &~ dicthiz,ldithiocarbarvlatr (5) Standard.. Stearic acid (I:. Merck, AG Darmstadt, (0.1723 g:;) in heptane.
71p7).
buffet
No. 67z), 6 ,uecluiv,Iml
PROCEllL-RE
Into a screw-cap 130.0 by 15.0 mm pipette 1.00 ml of substrate and 0.10 ml of serum. Mix, cap, and incubate for 30 min at 40’. Stop the reaction with 2.0 ml of copper reagent. Add 10.0 ml of chloroform and shake vigorously on the “shaker” for IO min. Centrifuge for 5 min and draw off the aqueous, blue-green layer with a water vacuum pump. Pipette into test tube chloroform layer 0.20 ml, chloroform 3.0 ml and diethyldithiocarbamate reagent 0.30 ml. Mix and after IO min read absorbance of the sample and of the serum-blank against reagents blank. Wavelength: 420.--450 nm (Hg 436 nm). Glass cuvette: r-cm light path. The serum blank is prepared by pipetting simultaneously 1.0 ml of substrate, 0.10 ml of serum, 2.0 mlof copperreagent and x0.0 ml of chloroform. The standard is prepared by, pipetting 0.50 ml of stearic acid in hep tane, 2.0 ml of copper reagent and 9.5 ml of chloroform. The reagents blank is prepared with all reagents alone (copper reagent, chloroform). The standard, the serum blank and the reagents blank are treated in the same way as the sample after incubation. A lipase unit is defined as the amount of enzyme which catalyses the hydrolysis of air olive oil substrate at 40’ to release I pequiv of fatty acid in I min. Serum lipase activity. is expressed in I.I-. Under the specific reaction conditions: I I.Ir.
=
I
Calculation:
~ecluiv~(r/30)-1~10000-1 Lipase activity
(in I.U.)
== 0.003 ;sequiv of stcaric =
acid.
SERUM
LIPASE
A sample--A A standard RESUL-I
S z\SD
201
DETERMINATIOh-
serum blank ~-. 1000
DISCUSSION
The kinetics of the enzymatic reaction has been studied by analyzing the enzyme content of a pool of human sera, at various dilutious with saline, to which pancreatic lipase was added. As shown in Fig. I, the enzyme activities lie on a straight line passing through the origin. Zero order kinectics is followed up to about 2200 I.U. In order to maintain the sensitivity of the method, it is very important to use in the preparation of the substrate solution an olive oil almost completely free from fatty acids.
2400 2200
2000 1800 16W 14w 1200 1000 8Ou 600 400 x10 0 140130
lx)
110
18
15
l’ig. I. Lipase activity (in I.U.) at various dilutions of a strum with very (WC test). Zero order kinetics are followed up to about zr~o 1.U. of lipasc.
The normal
values
of the proposed
method
high enzyme
were obtained
contents
by analyzing
220
samples of unselected sera, belonging to patients affected by different diseases. In all cases the amylase activity was tested in order to exclude eventual pancreatic diseases. The normal values are in the range o to 20 I.U. Only one pathologic serum (showing 296 Somogyi Units of amylase) gave with the present method an enzymatic content of ZIO I.U.; with the original technique of Cherry and Crandalll and with that of Tietz5 we got 6 and 3.2 Units respectively. ICEFERENCES I L j .+ 5 6 7
1. S. CHERRY AND L. A. CRAXDALL, Amer. ,I. Physml., IOO (1932) 266. I.. Smu.~ ASD P. DESXUELLE, Bzochim. Biopkys. Acta, 30 (1958) 513. 11. GLIcti ASD G. li. Rrs);Ix~, J. Bid. Chwz., IIO (1935) I*O. T. .\. JOHNSOS AND H. 1.. BOCKLYS, Avch. fntevnal Ned., 66 (1940) 62. S. 1%‘:. TIETZ, T. BORIIEX ANI) J. STAPLETOS, AWLEY. J. Clin. Pathol.. 31 (1959) IT. 13. SEIFFERT, Cltn. Chim. Acta, rX (1970) 5’. C. G. IKcmos ASI) D. SELIGSON, Amw. J. Clirz. Pnthol., 48 (1967) 307. Clzx. Chim.
.4&z,
143
37 (1972)
rq-roz
LIPPI et
202
H W. C. VOGEL Axis L. ZIEVE, Clin. Clwm., () (1963) 168. g W. G. DUNCOMBE, Biochenz. ,J., 88 (1963) 7. 10 R. J. HEKRY, Clinical Chcmistvy, Primzfilrs and Tcchnzcs,
Harper
p. ‘@I. 11 R. P. MACDOSALD AND Ii. 0. LE T“AVE, C~~IZ. C~PNZ., 3 (1962) 509. 12 J. H. T~OE ASD Ii. E. RI’LER, Anal. Iliochew., 6 (1963) 4;1. r3 PI’. 1%‘. TIETZ ANVD E. A. FIERECK, Cliv. Chiw. .4cta, 13 (1966) 3.52.
Clin. Chim.
ilcta,
37 (1972) 199-202
and
Row,
Iiwv
1.ak,
al.
rgfq,
CCA
199
4761
A RAPID
PHOTOMETRIC
MICROMETHOD
FOR
SERUM
LIPASE
DETERMINATION
U. LIPPI,
G. STEVI’INATO
Depavtmrnt (Received
0-f Pathology, Septcmbcr
I,
AND G. GliIDI
P. Cosma Hospdal,
35012 Camposampiero
(Italy)
1971)
A new micromethod for the determination of serum lipase activity is presented. Small quantities of serum are incubated for 30 min at 40’ in a substrate consisting of an olive oil suspension in the presence of deoxycholic acid. The pH is 8.5. The hydrolyzed fatty acids are determined with a photometric technique, with a slight modification of the method of Duncombe. The normal values are lower than 20 I.U.
INTRODCCTION
Several methods have been published for the determination of serum lipase, but none is fully satisfactory. Several suggested modifications of the Cherry-Crandalll method increase the sensitivity and reduce the time of incubation. Pancreatic lipase is able to hydrolyse esters of long chain fatty acids containing 8 to 18 carbon atoms. Therefore as substrate for lipase, esters of long chain fatty acids are to be preferred, but even triolein (olive oil) is hydrolyzed to a small extent by esterases. According
to Sarda
and Desnuelle2,
lipase
acts exclusively
on insoluble
sub-
strate. Fatty acids released by the hydrolysis of triglycerides by lipase are measured 6s7 techniques or by turbidimetrys. Several by titrimetic1T3p5 and spectrophotometric photometric methods are accurate and sensitive; however the use of very delicate and unstable substrates, an overlong incubation time and the sophisticated manipulations required to perform a lipase analysis preclude their use in many laboratories. A new photometric micromethod for serum lipase estimation is presented in this report. PRINCIPLE
Serum lipase hydrolyses olive oil triglycerides. One of the products of reaction are free fatty acids which are measured calorimetrically according to the method of Clin. Chim. Acta, 37 (1972) 199-~02
LIPPI
200
ct a/.
Duncombeg, with slight modifications. The substrate composition and the pH, the serum-substrate ratio, the incubation time and the effect of temperature have been evaluated by Henry’“, by Massion and Seligson7 and by several other workers”-I:$. Our present method is able to reduce the volume of serum (0.10 ml), and substrate (1.0 ml). Incubation time has been reduced to 30 min. The method is ver\r sensitive, accurate and it gives reproducible available from manufacturers+.
results. The reagents
are easil!, prepared
or in part
REAGESTS
(I) Substrate: Purified olive oil, 5 ml is homogenized (at o‘ in a I’otter~Elvelljcnl glass homogenizer fitted with a Teflon pestle) in IOO ml of a solution containing 0.242 g”/o of Tris-(hydroxymethyl)-aminomethane, 0.350 g:“,; of deoxycholic acid and of 0.02 go/, of ascorbic acid. The pH is adjusted to 8.50 with I X HCl or I ~‘v:?r’aOH. The emulsion can be used for 20~30 days if stored at +-lo. Olive oil is completely freed from fatty acids by passing it through alumina (I?. Merck, AC Darmstadt, So. (2) Chlorofon~, reagent grade. (PH , ‘fi:) Cofili,6~ reagmt: 0.27 Rl copper nitrate in 0.45 31 triethanolamine in butanol. (4) 9. o &~ dicthiz,ldithiocarbarvlatr (5) Standard.. Stearic acid (I:. Merck, AG Darmstadt, (0.1723 g:;) in heptane.
71p7).
buffet
No. 67z), 6 ,uecluiv,Iml
PROCEllL-RE
Into a screw-cap 130.0 by 15.0 mm pipette 1.00 ml of substrate and 0.10 ml of serum. Mix, cap, and incubate for 30 min at 40’. Stop the reaction with 2.0 ml of copper reagent. Add 10.0 ml of chloroform and shake vigorously on the “shaker” for IO min. Centrifuge for 5 min and draw off the aqueous, blue-green layer with a water vacuum pump. Pipette into test tube chloroform layer 0.20 ml, chloroform 3.0 ml and diethyldithiocarbamate reagent 0.30 ml. Mix and after IO min read absorbance of the sample and of the serum-blank against reagents blank. Wavelength: 420.--450 nm (Hg 436 nm). Glass cuvette: r-cm light path. The serum blank is prepared by pipetting simultaneously 1.0 ml of substrate, 0.10 ml of serum, 2.0 mlof copperreagent and x0.0 ml of chloroform. The standard is prepared by, pipetting 0.50 ml of stearic acid in hep tane, 2.0 ml of copper reagent and 9.5 ml of chloroform. The reagents blank is prepared with all reagents alone (copper reagent, chloroform). The standard, the serum blank and the reagents blank are treated in the same way as the sample after incubation. A lipase unit is defined as the amount of enzyme which catalyses the hydrolysis of air olive oil substrate at 40’ to release I pequiv of fatty acid in I min. Serum lipase activity. is expressed in I.I-. Under the specific reaction conditions: I I.Ir.
=
I
Calculation:
~ecluiv~(r/30)-1~10000-1 Lipase activity
(in I.U.)
== 0.003 ;sequiv of stcaric =
acid.
SERUM
LIPASE
A sample--A A standard RESUL-I
S z\SD
201
DETERMINATIOh-
serum blank ~-. 1000
DISCUSSION
The kinetics of the enzymatic reaction has been studied by analyzing the enzyme content of a pool of human sera, at various dilutious with saline, to which pancreatic lipase was added. As shown in Fig. I, the enzyme activities lie on a straight line passing through the origin. Zero order kinectics is followed up to about 2200 I.U. In order to maintain the sensitivity of the method, it is very important to use in the preparation of the substrate solution an olive oil almost completely free from fatty acids.
2400 2200
2000 1800 16W 14w 1200 1000 8Ou 600 400 x10 0 140130
lx)
110
18
15
l’ig. I. Lipase activity (in I.U.) at various dilutions of a strum with very (WC test). Zero order kinetics are followed up to about zr~o 1.U. of lipasc.
The normal
values
of the proposed
method
high enzyme
were obtained
contents
by analyzing
220
samples of unselected sera, belonging to patients affected by different diseases. In all cases the amylase activity was tested in order to exclude eventual pancreatic diseases. The normal values are in the range o to 20 I.U. Only one pathologic serum (showing 296 Somogyi Units of amylase) gave with the present method an enzymatic content of ZIO I.U.; with the original technique of Cherry and Crandalll and with that of Tietz5 we got 6 and 3.2 Units respectively. ICEFERENCES I L j .+ 5 6 7
1. S. CHERRY AND L. A. CRAXDALL, Amer. ,I. Physml., IOO (1932) 266. I.. Smu.~ ASD P. DESXUELLE, Bzochim. Biopkys. Acta, 30 (1958) 513. 11. GLIcti ASD G. li. Rrs);Ix~, J. Bid. Chwz., IIO (1935) I*O. T. .\. JOHNSOS AND H. 1.. BOCKLYS, Avch. fntevnal Ned., 66 (1940) 62. S. 1%‘:. TIETZ, T. BORIIEX ANI) J. STAPLETOS, AWLEY. J. Clin. Pathol.. 31 (1959) IT. 13. SEIFFERT, Cltn. Chim. Acta, rX (1970) 5’. C. G. IKcmos ASI) D. SELIGSON, Amw. J. Clirz. Pnthol., 48 (1967) 307. Clzx. Chim.
.4&z,
143
37 (1972)
rq-roz
LIPPI et
202
H W. C. VOGEL Axis L. ZIEVE, Clin. Clwm., () (1963) 168. g W. G. DUNCOMBE, Biochenz. ,J., 88 (1963) 7. 10 R. J. HEKRY, Clinical Chcmistvy, Primzfilrs and Tcchnzcs,
Harper
p. ‘@I. 11 R. P. MACDOSALD AND Ii. 0. LE T“AVE, C~~IZ. C~PNZ., 3 (1962) 509. 12 J. H. T~OE ASD Ii. E. RI’LER, Anal. Iliochew., 6 (1963) 4;1. r3 PI’. 1%‘. TIETZ ANVD E. A. FIERECK, Cliv. Chiw. .4cta, 13 (1966) 3.52.
Clin. Chim.
ilcta,
37 (1972) 199-202
and
Row,
Iiwv
1.ak,
al.
rgfq,