- Email: [email protected]
Nephelometric determination of pancreatic enzymes
Clilzica Chimica Acta, 43 (1973) ((3 Elscvier Scientific Publishing
5-12 Company,
Amsterdam
- Printed
in The Netherlands
5
cc4 5412
NEPHELOMETRIC
DETERMINATION
OF
PANCREATIC
ENZYMES
I. i\hIYI,.4SE
SUMMARY
A nephelometric procedure for the kinetic analysis of amylase has been developed. A quantitative result may be obtained in z to 4 min with 50 ,~l or less of serum or urine. Hydrolysis of the substrate results in reduction of light scattering which is measured continuously. The reaction follows zero order kinetics. Enzyme activity is determined from the slope of the line traced by a chart recorder. Formazin is used as a reproducible turbidity standard. ,4 reproducible preparation of amylopectin is used as the substrate.
INTRODUCTION
Classical methods for determination of amylase and their more recent modifications generally employ the use of soluble starch substrates and relate the formation of reducing sugarslpd or the decrease in iodine-stainable starch5 to amylase activity. Starchdye complexes have also been developed as substrates6~7. Turbidimetric methods have been described81g which relate a decrease in absorbance of a turbid starch substrate to enzyme activity. These have the advantage of simplicity and rapidity while maintaining high specificity. However, this technique has suffered from a lack of adequate standards*, from poor precision at near normal levels of amylase”‘, and from inadequate substrate stability. The method described in this paper utilizes nephelometry to measure the decrease in light scattering of an amylopectin substrate resulting from amylase activity. Kephelometry measures light scattered by a turbid suspension and is a more sensitive measurement of turbidity than is absorption photometry. The nephelometric measurement correlates linearly with turbidity up to a point determined by the level of turbidity and is a function of intensity of incident light. The proposed methods involves a kinetic measurement, in that it measures decrease in turbidity continuously. Enzyme activity is proportional to the recorded slope. The enzyme concentration is related to the rate of decrease of turbidity. The amount of light scattering-under the
6
ZINTERHOFER
controlled conditions can be established with turbidity secondary enzyme standards. The ease of measuring small changes of turbidity
standards,
thereby
nephelometrically
Pt d.
avoiding allows for
measurement of low amylase levels found in the normal range. Using 50 ,uI or less of serum or urine one may obtain a quantitative result in 2 to 5 min. Since amvlase measurements are most frequently made on an emergency basis, this short but complete quantitative analysis has great value in a hospital. This method also provides a base for automating the amylase procedure. MATERIALS
AXD
METHODS
Any nephelometer (or fluorometer used as a nephelometer), equipped with a chart recorder with both incremental and continuous sensitivity controls, and with a control for offset over the entire range at any selected sensitivity may be used. Temperature control is maintained by pumping water from a 30” water bath through a water-jacketed cuvette. The use of higher wavelengths (600 to 1000 nm) is preferable since such light is more effectively scattered by larger particles and newer photometric detectors work well in this region. Preliminary studies were performed on a recording spectrofluorometer (PerkinElmer model 204)‘“. The nephelometer used for most of these experiments n-as constructed in this laboratory. The body, an aluminum block measuring approximatel! 4 x I x z inches, is drilled lengthwise around the periphery to form channels for circulating water from a water bath. A z-ml round thin glass tube (II mm dia.), with outlets at top and bottom is used as the cuvette. The cuvettc is connected to a 3-1111 syringe so that it can be filled by aspiration. A lens-ended lamp (GE ;i! 252) illuminates the sample and a P.I.N. diode photodetector (Monsanto JID-z) set at right angles to the is scattered 1~~.the sample but inlight path detects scattered light. “White-light” asmuch as the diode photodetector’s sensitivity lies largely in the red and near infrared region, the system effectivelv measures light scatter in the 600o1000 mn range. The lamp is powered by a precision-regulated 2.4 (* o.0176) power supply. The current output from the photodetector is fed into a PET input, operational amplifier (Bell and Howell DDCooS), and the output from this is fed into scaling circuitry. The recording instrument is an Esterline Minigraph fitted with a chart speed drive of j”,Imin. The full scale deflection can be set from 5 V/inch to 0.5 17/inch. Provision was made to null out any desired level of baseline voltage so that the full sensitivity might be employed at any voltage output. Reagents Chemicals
are reagent grade. Avoid contamination with dust. Formazin turbidity stock standard 11,12: Dissolve 0.5 g hydrazine sulfate’“* and 5.0 g hexamethylenetetramine :Zi G:8 in separate Ioo-ml volumetric flasks, each containing approximately 40 ml water. Quantitatively transfer the contents of one flask to the other, mix and adjust to IOO ml. Allow to stand at room temperature for 48 11. The turbiditv of this * Perkin-Elmer Corp., Norwalk, Connecticut. ** Eastman Kodak Co., Rochester, New York. * * * J. T. Raker Co., Phillipsburg, Xew Jersey.
PANCREATIC ENZYME DETERMINATION
7
stock formazin is reproducible to + 1% and stable for 3 to 6 months. 4000 formazin turbidity units (4000 FTU).
It is defined as
Formazin dilution: Mix formazin stock standard well and make a I : 20 dilution in water. This provides a turbidity of 200 FTU and is stable for at least two days. Preparation of stock substrate: Prepare diluent by dissolving 18.12 g Tris (hydroxymethyl)amino methane and 1.750 g sodium chloride per liter of water. Weigh 0.8 g amylopcctin (amylose-free) * into Ioo-ml volumetric flask. Prepare a 95” water bath on a hot plate with a built-in magnetic stirrer. Place a magnetic stirring bar in the flask and add 4 ml dimethylsulfoxide (DMSO). Mix immediately until the amylopectin is completely dispersed then place the flask in the 95” water bath. Heat and stir the mixture until completely clear (I to z min) except for entrapped bubbles. Slowly add 80 ml diluent with continuous stirring. Allow temperature to again reach 95”. Place in 30” water bath, allow to cool and add 5 ml 0.02 g/ml sodium azide. Remove stirring bar (with another magnet) and dilute to IOO ml with water. This stock substrate is kept in a tightly covered amber bottle in the 30’ water bath at all times. Avoid contamination of this substrate with dirty pipettes or saliva. Working substrate: Make up substrate daily or as needed by adding about 0.5 ml 0.5 M HCl per 2.5 ml stock substrate in order to establish the pH between 7.1 to 7.3.
Standardization of nefihelometer: The 200 FTU standard is in the range of the level of light dispersion produced by the working substrate and is used to calibrate the response of the nephelometer. Zero the instrument with a water blank then place the 200 FTU standard into the nephelometer cuvette and adjust the continuous sensitivity setting so that a 2/3 full scale reading is obtained on the recorder. The nephelometer is thereby calibrated. This calibration is checked and readjusted periodically in case of instrument drift. A daily check should be satisfactory if the nephelometer is stable. Measurement of serum OYurine amylase: Pipette 3-ml aliquots of working substrate into clean, dust-free tubes in a 30’ water bath. Add 50 ~1 serum or urine to a 3-ml aliquot, mix well and transfer to the fibrin particles or urinary sediment as large noise. Lipemic sera produce little noise. Set IO times the level at which 200 FTU gave a control as necessary. activity is expressed
Follow the reaction on the recorder for 2 to 4 minutes. Enzyme as the decrease in light scattering in FTU per min, per ml of
sample under the defined conditions. 2
x ‘7
200 = FTU
nephelometer cuvette. Avoid picking up suspended particulate matter will produce the incremental sensitivity control to 5 or z/3 full scale reading and adjust the off-set
This is calculated
as follows:
x G = FTU/min/ml
of nephelometric
standard for 200 FTU 4R = Change in scale units during enzyme measurement. factor must be included in establishing 4R. t = time in minutes over which ,4R is measured V = volume in ml of sample
RS x Scale units on nephelometer
* Calbiochem.
J,os
rlngcles,
California
Note that scale expansion
ZINTEKHOFEK
8
et d.
Since the instrument is always calibrated in the same manner, a single factor can bc used which converts scale divisions change per selected time per 50 ~1 sample to FTU/min/ml. It the level is greater than 350 FTU/min/ml, use only zo ~tl of serum or urine or use a dilution of the specimen. Analyze a control serum each dav as a check on sub strate stability. The substrate is discarded after 7 days or when a change in the amvlase activity of the control serum occurs. RESCLTS
Formazin
standardization
The stock formazin standard remained nephelometrically stable throughout a 4-month experimental period. Dilutions to 200 FTC made from five different stock formazin suspensions varied within -+ I(!/;, and were stable for at least two davs. Stability of substvatc The substrate (different batches
made up on different
days) was reproducible
to
within & 3% when evaluated with the same control serum. Any single batch of substrate was stable to within :fI 3 O/ /,, for a period of about 7 da\-s. Thereafter a slow dccrease in the absolute turbidity was accompanied by decreased activity with control serums. Occasionally slight deterioration occurred before this time ; this was detected by daily analysis of control serum. Reaction co?zditio?u $H. The pH of the stock substrate was adjusted with HCl over a pH range of 6.4 to 8.5. The reaction rate was optimal and constant between a pH of 7.0 and 7.4 at 30’ as shown in Fig. I.
6.4
6.8
7.2 PH
7.6
8.0
Temfierature. The effect of temperature is shown in Fig. 2. A temperature of 30” was chosen for this method. Enzyme cowxntratioa. The effect of enzyme concentration was demonstrated b! analyzing- a progressively diluted serum with high amylase activity. The resulting rcaction rate was linear with enzyme concentration (Fig. 3) to a level of about 350 FTU/min/ml. At higher enzyme levels, the linearity did not persist over a 4-min
PANCREATIC
I
ENZYME
DETERMINATIOK
1
,
25 Fig. 2. Jiffcct
of temperature
I:1616
on reaction
I.4 I:3
of arnylase
I
40
45
rate.
I:2
Serum Fig. 3. Effect
.
30 35 Temperature CC)
9
Dtlutions
concentration
on reaction
rate.
period. The reaction followed zero order kinetics for at least 4 min when the enzyme level was below 350 FTU/min/ml. Fig. 4 illustrates the continuous recorder tracings of nephelometric amylase analyses. Precision The standard deviation of the method determined by running duplicate samples (mean IOZ FTU/min/ml) was 2.8 FTU/min/ml.
17 pairs
of
Comj5arison with standard method Xmylase activity in 50 samples of serum and urine was measured by both the iodometric method of Smith and Roe” and by the proposed method (Fig. 5). The methods were linearly related with a correlation coefficient of 0.93. The regression curve indicates that the amylase activity expressed in FTU/min/ml is approximately half that when expressed as Somogyi units per IOO ml. 2:ormal range The amylase activity of 32 serums from normal blood donors was measured. They averaged 46 FTU/min/ml and ranged from 18 to 75 FTU/min/ml with a S.D. of 13.
IO
t?tat.
ZIXTERHOFER
400 Amylose
800 (Somogl
1200 unit4
1600
Fig. .+. Continuous recordings from four strum amylasc mcasurcmcnts. The lines have been drawn parallel with the recorded points to show the original tracing. The drawn lines (usually through the recorded points) are used to establish the change in turbidity for calculation of amylase activity. Time is on the ordinate and voltage (proportional to turbidity or substrate) is on the abscissa. The bottom curve (rgco FTIJ) xvas measured with 20 1~1of serum rather than so. Kormally this serum would lx diluted further. Fig. 5. Comparison of serum nmylase activity an iodomctric method (abscissa).
as measured by the proposed method (ordinate)
with
The amylase activity of fourteen 24-h urine specimens from normal volunteers ranged from 83500 to 354000 FTU/min/total volume with mean of 194500PTU/minj total volume. Interferences The presence of dialyzable inhibitors in urine was ruled out by measuring the amylase in 5 urines before and after 18 h of continuous dialysis. No change in activity occurred. Com$arison with lifiase A serum lipase was performed by the Cherry-Crandallrb method on 18 sera from patients with an elevated amylase by the proposed nephelometric procedure. Fifteen of these demonstrated an elevated lipase (> than 1.5 units).
PAh‘CREATIC
ENZYME
DETERMINATION
II
DISCUSSION
This nephelometric approach to amylase determination has the advantage of simplicity, speed and good precision. The analytical result is unaffected by color or turbidity of the sample since only a change that occurs in light dispersion of the substrate is measured. Turbidimetric measurements of amylase activity have been considered specific (refs 8, 9). Measurements of turbidity changes by nephelometry are more sensitive than by absorptiometry which permits shorter analysis time. Measurement of the substrate directly, as is done here, may be more specific than the measurement of poorly defined reaction products. A complete amylase measurement is made in a single step by adding a micro portion of sample to the substrate and recording the reaction in the nephelometer over a z- to 4-min period. The activity is calculated from the slope of the recorded line in FTU/min/ml and is the unit of amylase activity used here. The short time required and the simplicity of the technique permit it to be easily automated. This type of measurement is particularly suitable for emergency service. The method is standardized by (a) calibration of the nephelometer response with a formazin turbidity standard and (b) by the use of a substrate with reproducible and stable response to amylase activity as measured nephelometrically. Formazin is a stable, reproducible, turbidity standard which has been previously describedll and has been used in industry for this purposel2. When prepared by the method described, it produces a turbidity which by definition equals 4000 formazin turbidity units (formerly 4000 Jackson turbidity units)l”. An aqueous dilution is made to a level of turbidity within the working range for enzyme analysis. Substrates used for most previous amylase methods were found by us to be nephelometricallp unstable and variable. Sensitivity variation, and flocculation of these starch suspensions occurred. The possibility of dispersing amylopectin in dimethylsulfoxide, as suggested by Friedlander and Berk4, was explored and found to produce complete dispersion and prevent flocculation if the two were in proper relative concentration. However, when this initial substrate (8 g/l amyopectin, 80 ml/l DMSO in pH 7.0, 0.1 ibZ Tris buffer) was refrigerated, change in dispersion of the amylopectin occurred which was apparent by increased turbidity and altered response to enzyme controls. This change was reversible after returning the substrate to 30’ for several hours. If the substrate was continuously maintained at 3o”, it remained stable initially then showed a decrease in turbidity and response to amylase within 24 to 72 h. However, this deterioration was postponed for 7 days if the substrate was kept at pH IO as described above. As a result of this experience, the stock substrate is maintained at 30’ and pH IO at all times. A pH of 7.1 to 7.2 is achieved by adding 1.0 ml of 0.5 RI HCl to 5.0 ml of stock substrate. Sodium azide which was added as a preservative did not influence activity at the concentration used. The concentration of amylopectin used was sufficient to measure up to 350 FTU/ ruin/ml amylase (700 Somogyi units) in 50 ~1 serum or urine without loss of linearity. 0.1 JI Tris buffered the serum substrate mixture adequately at this pH although it is far from the optimal buffering range of Tris. The substrate stability was greater than that achieved with phosphate buffer. The values obtained by this method are linearly related to those obtained by an
ZIKTEKHOFEK
12
et d.
iodometric methodj. The comparison may have suffered from the limits of reproducibility of the starch-iodine method which has a coefficient of variation in tire range of zoq/, (ref. 13). Enzyme activity
is expressed
as FTLJ/ min/ml. The study
of normals
and tire
comparison to the iodometric method indicate that I FTU/min/ml is equal to approximately two Somog+ units/roe ml (see Fig. 5). The finding of an elevated lipase (Cherry-Crandall) in 15 of 18 patients witlr an elevated amylase (nephelometric)
supports the clinical validity of theproposcd
method.
5-12 Company,
Amsterdam
- Printed
in The Netherlands
5
cc4 5412
NEPHELOMETRIC
DETERMINATION
OF
PANCREATIC
ENZYMES
I. i\hIYI,.4SE
SUMMARY
A nephelometric procedure for the kinetic analysis of amylase has been developed. A quantitative result may be obtained in z to 4 min with 50 ,~l or less of serum or urine. Hydrolysis of the substrate results in reduction of light scattering which is measured continuously. The reaction follows zero order kinetics. Enzyme activity is determined from the slope of the line traced by a chart recorder. Formazin is used as a reproducible turbidity standard. ,4 reproducible preparation of amylopectin is used as the substrate.
INTRODUCTION
Classical methods for determination of amylase and their more recent modifications generally employ the use of soluble starch substrates and relate the formation of reducing sugarslpd or the decrease in iodine-stainable starch5 to amylase activity. Starchdye complexes have also been developed as substrates6~7. Turbidimetric methods have been described81g which relate a decrease in absorbance of a turbid starch substrate to enzyme activity. These have the advantage of simplicity and rapidity while maintaining high specificity. However, this technique has suffered from a lack of adequate standards*, from poor precision at near normal levels of amylase”‘, and from inadequate substrate stability. The method described in this paper utilizes nephelometry to measure the decrease in light scattering of an amylopectin substrate resulting from amylase activity. Kephelometry measures light scattered by a turbid suspension and is a more sensitive measurement of turbidity than is absorption photometry. The nephelometric measurement correlates linearly with turbidity up to a point determined by the level of turbidity and is a function of intensity of incident light. The proposed methods involves a kinetic measurement, in that it measures decrease in turbidity continuously. Enzyme activity is proportional to the recorded slope. The enzyme concentration is related to the rate of decrease of turbidity. The amount of light scattering-under the
6
ZINTERHOFER
controlled conditions can be established with turbidity secondary enzyme standards. The ease of measuring small changes of turbidity
standards,
thereby
nephelometrically
Pt d.
avoiding allows for
measurement of low amylase levels found in the normal range. Using 50 ,uI or less of serum or urine one may obtain a quantitative result in 2 to 5 min. Since amvlase measurements are most frequently made on an emergency basis, this short but complete quantitative analysis has great value in a hospital. This method also provides a base for automating the amylase procedure. MATERIALS
AXD
METHODS
Any nephelometer (or fluorometer used as a nephelometer), equipped with a chart recorder with both incremental and continuous sensitivity controls, and with a control for offset over the entire range at any selected sensitivity may be used. Temperature control is maintained by pumping water from a 30” water bath through a water-jacketed cuvette. The use of higher wavelengths (600 to 1000 nm) is preferable since such light is more effectively scattered by larger particles and newer photometric detectors work well in this region. Preliminary studies were performed on a recording spectrofluorometer (PerkinElmer model 204)‘“. The nephelometer used for most of these experiments n-as constructed in this laboratory. The body, an aluminum block measuring approximatel! 4 x I x z inches, is drilled lengthwise around the periphery to form channels for circulating water from a water bath. A z-ml round thin glass tube (II mm dia.), with outlets at top and bottom is used as the cuvette. The cuvettc is connected to a 3-1111 syringe so that it can be filled by aspiration. A lens-ended lamp (GE ;i! 252) illuminates the sample and a P.I.N. diode photodetector (Monsanto JID-z) set at right angles to the is scattered 1~~.the sample but inlight path detects scattered light. “White-light” asmuch as the diode photodetector’s sensitivity lies largely in the red and near infrared region, the system effectivelv measures light scatter in the 600o1000 mn range. The lamp is powered by a precision-regulated 2.4 (* o.0176) power supply. The current output from the photodetector is fed into a PET input, operational amplifier (Bell and Howell DDCooS), and the output from this is fed into scaling circuitry. The recording instrument is an Esterline Minigraph fitted with a chart speed drive of j”,Imin. The full scale deflection can be set from 5 V/inch to 0.5 17/inch. Provision was made to null out any desired level of baseline voltage so that the full sensitivity might be employed at any voltage output. Reagents Chemicals
are reagent grade. Avoid contamination with dust. Formazin turbidity stock standard 11,12: Dissolve 0.5 g hydrazine sulfate’“* and 5.0 g hexamethylenetetramine :Zi G:8 in separate Ioo-ml volumetric flasks, each containing approximately 40 ml water. Quantitatively transfer the contents of one flask to the other, mix and adjust to IOO ml. Allow to stand at room temperature for 48 11. The turbiditv of this * Perkin-Elmer Corp., Norwalk, Connecticut. ** Eastman Kodak Co., Rochester, New York. * * * J. T. Raker Co., Phillipsburg, Xew Jersey.
PANCREATIC ENZYME DETERMINATION
7
stock formazin is reproducible to + 1% and stable for 3 to 6 months. 4000 formazin turbidity units (4000 FTU).
It is defined as
Formazin dilution: Mix formazin stock standard well and make a I : 20 dilution in water. This provides a turbidity of 200 FTU and is stable for at least two days. Preparation of stock substrate: Prepare diluent by dissolving 18.12 g Tris (hydroxymethyl)amino methane and 1.750 g sodium chloride per liter of water. Weigh 0.8 g amylopcctin (amylose-free) * into Ioo-ml volumetric flask. Prepare a 95” water bath on a hot plate with a built-in magnetic stirrer. Place a magnetic stirring bar in the flask and add 4 ml dimethylsulfoxide (DMSO). Mix immediately until the amylopectin is completely dispersed then place the flask in the 95” water bath. Heat and stir the mixture until completely clear (I to z min) except for entrapped bubbles. Slowly add 80 ml diluent with continuous stirring. Allow temperature to again reach 95”. Place in 30” water bath, allow to cool and add 5 ml 0.02 g/ml sodium azide. Remove stirring bar (with another magnet) and dilute to IOO ml with water. This stock substrate is kept in a tightly covered amber bottle in the 30’ water bath at all times. Avoid contamination of this substrate with dirty pipettes or saliva. Working substrate: Make up substrate daily or as needed by adding about 0.5 ml 0.5 M HCl per 2.5 ml stock substrate in order to establish the pH between 7.1 to 7.3.
Standardization of nefihelometer: The 200 FTU standard is in the range of the level of light dispersion produced by the working substrate and is used to calibrate the response of the nephelometer. Zero the instrument with a water blank then place the 200 FTU standard into the nephelometer cuvette and adjust the continuous sensitivity setting so that a 2/3 full scale reading is obtained on the recorder. The nephelometer is thereby calibrated. This calibration is checked and readjusted periodically in case of instrument drift. A daily check should be satisfactory if the nephelometer is stable. Measurement of serum OYurine amylase: Pipette 3-ml aliquots of working substrate into clean, dust-free tubes in a 30’ water bath. Add 50 ~1 serum or urine to a 3-ml aliquot, mix well and transfer to the fibrin particles or urinary sediment as large noise. Lipemic sera produce little noise. Set IO times the level at which 200 FTU gave a control as necessary. activity is expressed
Follow the reaction on the recorder for 2 to 4 minutes. Enzyme as the decrease in light scattering in FTU per min, per ml of
sample under the defined conditions. 2
x ‘7
200 = FTU
nephelometer cuvette. Avoid picking up suspended particulate matter will produce the incremental sensitivity control to 5 or z/3 full scale reading and adjust the off-set
This is calculated
as follows:
x G = FTU/min/ml
of nephelometric
standard for 200 FTU 4R = Change in scale units during enzyme measurement. factor must be included in establishing 4R. t = time in minutes over which ,4R is measured V = volume in ml of sample
RS x Scale units on nephelometer
* Calbiochem.
J,os
rlngcles,
California
Note that scale expansion
ZINTEKHOFEK
8
et d.
Since the instrument is always calibrated in the same manner, a single factor can bc used which converts scale divisions change per selected time per 50 ~1 sample to FTU/min/ml. It the level is greater than 350 FTU/min/ml, use only zo ~tl of serum or urine or use a dilution of the specimen. Analyze a control serum each dav as a check on sub strate stability. The substrate is discarded after 7 days or when a change in the amvlase activity of the control serum occurs. RESCLTS
Formazin
standardization
The stock formazin standard remained nephelometrically stable throughout a 4-month experimental period. Dilutions to 200 FTC made from five different stock formazin suspensions varied within -+ I(!/;, and were stable for at least two davs. Stability of substvatc The substrate (different batches
made up on different
days) was reproducible
to
within & 3% when evaluated with the same control serum. Any single batch of substrate was stable to within :fI 3 O/ /,, for a period of about 7 da\-s. Thereafter a slow dccrease in the absolute turbidity was accompanied by decreased activity with control serums. Occasionally slight deterioration occurred before this time ; this was detected by daily analysis of control serum. Reaction co?zditio?u $H. The pH of the stock substrate was adjusted with HCl over a pH range of 6.4 to 8.5. The reaction rate was optimal and constant between a pH of 7.0 and 7.4 at 30’ as shown in Fig. I.
6.4
6.8
7.2 PH
7.6
8.0
Temfierature. The effect of temperature is shown in Fig. 2. A temperature of 30” was chosen for this method. Enzyme cowxntratioa. The effect of enzyme concentration was demonstrated b! analyzing- a progressively diluted serum with high amylase activity. The resulting rcaction rate was linear with enzyme concentration (Fig. 3) to a level of about 350 FTU/min/ml. At higher enzyme levels, the linearity did not persist over a 4-min
PANCREATIC
I
ENZYME
DETERMINATIOK
1
,
25 Fig. 2. Jiffcct
of temperature
I:1616
on reaction
I.4 I:3
of arnylase
I
40
45
rate.
I:2
Serum Fig. 3. Effect
.
30 35 Temperature CC)
9
Dtlutions
concentration
on reaction
rate.
period. The reaction followed zero order kinetics for at least 4 min when the enzyme level was below 350 FTU/min/ml. Fig. 4 illustrates the continuous recorder tracings of nephelometric amylase analyses. Precision The standard deviation of the method determined by running duplicate samples (mean IOZ FTU/min/ml) was 2.8 FTU/min/ml.
17 pairs
of
Comj5arison with standard method Xmylase activity in 50 samples of serum and urine was measured by both the iodometric method of Smith and Roe” and by the proposed method (Fig. 5). The methods were linearly related with a correlation coefficient of 0.93. The regression curve indicates that the amylase activity expressed in FTU/min/ml is approximately half that when expressed as Somogyi units per IOO ml. 2:ormal range The amylase activity of 32 serums from normal blood donors was measured. They averaged 46 FTU/min/ml and ranged from 18 to 75 FTU/min/ml with a S.D. of 13.
IO
t?tat.
ZIXTERHOFER
400 Amylose
800 (Somogl
1200 unit4
1600
Fig. .+. Continuous recordings from four strum amylasc mcasurcmcnts. The lines have been drawn parallel with the recorded points to show the original tracing. The drawn lines (usually through the recorded points) are used to establish the change in turbidity for calculation of amylase activity. Time is on the ordinate and voltage (proportional to turbidity or substrate) is on the abscissa. The bottom curve (rgco FTIJ) xvas measured with 20 1~1of serum rather than so. Kormally this serum would lx diluted further. Fig. 5. Comparison of serum nmylase activity an iodomctric method (abscissa).
as measured by the proposed method (ordinate)
with
The amylase activity of fourteen 24-h urine specimens from normal volunteers ranged from 83500 to 354000 FTU/min/total volume with mean of 194500PTU/minj total volume. Interferences The presence of dialyzable inhibitors in urine was ruled out by measuring the amylase in 5 urines before and after 18 h of continuous dialysis. No change in activity occurred. Com$arison with lifiase A serum lipase was performed by the Cherry-Crandallrb method on 18 sera from patients with an elevated amylase by the proposed nephelometric procedure. Fifteen of these demonstrated an elevated lipase (> than 1.5 units).
PAh‘CREATIC
ENZYME
DETERMINATION
II
DISCUSSION
This nephelometric approach to amylase determination has the advantage of simplicity, speed and good precision. The analytical result is unaffected by color or turbidity of the sample since only a change that occurs in light dispersion of the substrate is measured. Turbidimetric measurements of amylase activity have been considered specific (refs 8, 9). Measurements of turbidity changes by nephelometry are more sensitive than by absorptiometry which permits shorter analysis time. Measurement of the substrate directly, as is done here, may be more specific than the measurement of poorly defined reaction products. A complete amylase measurement is made in a single step by adding a micro portion of sample to the substrate and recording the reaction in the nephelometer over a z- to 4-min period. The activity is calculated from the slope of the recorded line in FTU/min/ml and is the unit of amylase activity used here. The short time required and the simplicity of the technique permit it to be easily automated. This type of measurement is particularly suitable for emergency service. The method is standardized by (a) calibration of the nephelometer response with a formazin turbidity standard and (b) by the use of a substrate with reproducible and stable response to amylase activity as measured nephelometrically. Formazin is a stable, reproducible, turbidity standard which has been previously describedll and has been used in industry for this purposel2. When prepared by the method described, it produces a turbidity which by definition equals 4000 formazin turbidity units (formerly 4000 Jackson turbidity units)l”. An aqueous dilution is made to a level of turbidity within the working range for enzyme analysis. Substrates used for most previous amylase methods were found by us to be nephelometricallp unstable and variable. Sensitivity variation, and flocculation of these starch suspensions occurred. The possibility of dispersing amylopectin in dimethylsulfoxide, as suggested by Friedlander and Berk4, was explored and found to produce complete dispersion and prevent flocculation if the two were in proper relative concentration. However, when this initial substrate (8 g/l amyopectin, 80 ml/l DMSO in pH 7.0, 0.1 ibZ Tris buffer) was refrigerated, change in dispersion of the amylopectin occurred which was apparent by increased turbidity and altered response to enzyme controls. This change was reversible after returning the substrate to 30’ for several hours. If the substrate was continuously maintained at 3o”, it remained stable initially then showed a decrease in turbidity and response to amylase within 24 to 72 h. However, this deterioration was postponed for 7 days if the substrate was kept at pH IO as described above. As a result of this experience, the stock substrate is maintained at 30’ and pH IO at all times. A pH of 7.1 to 7.2 is achieved by adding 1.0 ml of 0.5 RI HCl to 5.0 ml of stock substrate. Sodium azide which was added as a preservative did not influence activity at the concentration used. The concentration of amylopectin used was sufficient to measure up to 350 FTU/ ruin/ml amylase (700 Somogyi units) in 50 ~1 serum or urine without loss of linearity. 0.1 JI Tris buffered the serum substrate mixture adequately at this pH although it is far from the optimal buffering range of Tris. The substrate stability was greater than that achieved with phosphate buffer. The values obtained by this method are linearly related to those obtained by an
ZIKTEKHOFEK
12
et d.
iodometric methodj. The comparison may have suffered from the limits of reproducibility of the starch-iodine method which has a coefficient of variation in tire range of zoq/, (ref. 13). Enzyme activity
is expressed
as FTLJ/ min/ml. The study
of normals
and tire
comparison to the iodometric method indicate that I FTU/min/ml is equal to approximately two Somog+ units/roe ml (see Fig. 5). The finding of an elevated lipase (Cherry-Crandall) in 15 of 18 patients witlr an elevated amylase (nephelometric)
supports the clinical validity of theproposcd
method.