Determination ok amylase activity in biological fluids

(‘I~INIC‘.~ (‘HIMI(‘4

250

I)E’l-ERMINATION

.\Cf.\

\-01..

01; AMYLASE A~:‘HVIT\’ IN BIOI,O(;I(‘.41~ I;Ll-11)s

For many years prior to the commencement

of the tollowing

method used in our laboratories for determination of amylase that of !%MOGYI I, modified to employ H.UWING’S modification Hartman

method

1 (rc).joi

for estimation

of sugar as described

by

investigations,

the

activity in serum was 2, :I of the Schaffcr and

KING

I.

Howc\rcr. during

certain studies concerned with disease of the pancreas, it became necessary to car-r\. out daily a large number of determinations of amylase activity on both serum and nrinc. For this purpose WC found that our modification time-consuming.

The method

differs from the Somogyi

of

NOIC~Y

5,

of

which also

SOMOGYI’S

starch

LWS

method only in the use of alkaline

First of all, it appeared

but is actually

a mixture

to us that since starch

of two carbohydrates,

amylose

as substrate

ferricvanide

alkaline copper tartratc, was also too lengthy a proccdurc. ‘4part from this objection, WC felt that the use of starch to criticism.

method I was too and

in place of

as substrate

MYLSopru

is not a single compound

and amylopectin,

and since

the proportion of these (about zocj;, amylose and Xo’$,, amylopectin in potato starch) varies not only with the source of the starch but also with different batches from the same source, its USC as substrate for amvlasc might not give rc~produciblc results. Furthermore,

KIIW

ANL)

HOPKINS

6 lu\;c

shown

that

salivary,

amylase

(ptyalin)

liberates much maltose from amylose and very little from amylopectin. These workers have also shown that as hydrolysis proceeds and the substrate chains become shorter. the fission products

contain

an increasing

with the results of other WJrkN3 that glucose makes an earlier

proportion

of glucose. This is in accordancr

(bfYIw~~~.Ic i, .4t3Ih. .ANII (‘:~I,I)wi~I.I~ “) who

appearance

in the cnzy mic hydrolysis

also showed of starch or

amy.lopectin than of amylosc. Whilst it was realiscd that tllesc conclusions ha\-c IXYW drawn from work carried out with malt rl-amylase, salivary. cl-amylase and the CL-and /,‘-amylases of certain bacteria and that it may bc unwise to apply these results to serum amylasc, it was felt that for our purposes WC would ha\-e to exclude an>. method which uses starch as substrate. I~urthcrmorc, we would hale to cxcludc methods based on the determination of the reducing power of the h!,drolysis products of polysaccharidc substrates. Other substrates which have been used include glycogen (Sont~~ .\NI) ~,I\.EKs “) and amylosc (HIDET Srrc;l: Fr;w;\ 1”). The turbidimetric method of PEIMI.~A AKII REINHOI,L)‘~, and the method of SCOTT ANI) MELVIN 12 using dcxtran as substrate havt recently been brought to our notice. The anthrone reagent used in the determination of the glycogen hydrolysatc is unstable. ~‘AKROLL ANI) V.%N I)YK ‘ESl~tvc determined the activity of cr-amylasc by an adsorption indicator. In this method enzymic hydrolysis of starch is followed by addition of congo red which is adsorbed by the starch.

VOL. 1 (1956)

AMYLASEACTIVITY IN BIOLOGICALFLUIDS

257

Subsequent addition of iodine allows reaction between the halogen and the iodinestaining products, but reaction with starch is impeded by the dye. Methods using calorimetry follouing addition of iodine but with starch as substrate have been described by HIJGGINS AND RUSSELL I*, SMITH AND ROE Is, TELLER 16, and VAN LOON AND LIKINS~‘. It was decided, therefore, in view of the apparent shortcoming of other methods to investigate the use of amylose as substrate. As a starting point, we chose the coloriof the metric method which HOBSON AND MACPHERSON18 devised for determination activity of a-amylase of rumen bacteria, and modified this as necessary so that it would be applicable to blood serum and to urine. The choice of a method employing measurement of the optical density of an amylose-iodine complex appeared to be justified from the findings of MOULD AND SYNGE [email protected] workers have shown that the hydrolysis products of amylose as complexes with iodine-iodide may be separated by continuous electrophoresis into three differently coloured zones. Further, by application of the method of MOULD AND SYNGE 2othey were able to show by electrokinetic ultrafiltration in collodion membranes that the colour of the zone was related to molecular chain length. The electrophoretic fractions consisted of a blue zone containing compounds with a spread of degree of polymerisation (DP) of 40 to 130, a red zone of DP ~5 to 40 and an orange zone of DP IO to 25. In addition to these zones, there was a further zone of DP < IO which scarcely stained at all. The presence of an achroic fraction of DP < IO was demonstrated by paper chromatography. MOULD AND SYNGE I9 have also shown that the ratio of blue-to-red-staining material formed during a-amylase hydrolysis of amylose is comparatively high even when a considerable degree of hydrolysis has occurred. Hence it would appear that measurement of the optical density of the bluestaining hydrolysate produced by action of amylase on amylose might form the basis of an indirect method for determination of amylase activity. Furthermore, such a method would be likely to give more accurate results if hydrolysis were stopped at a stage where the concentration of the red-staining material was so low as to be negligible. Under such conditions, it was hoped that there would be a linear relationship between the amount of enzyme present and the decrease in optical density of the bluestaining hydrolysate. That this is so over a wide range of amylase activity has been established and details are given below. If hydrolysis is allowed to proceed beyond this stage the concentration of red-staining dextrin (i.e. of DP 25 to 40) increases and addition of iodine-iodide yields a purple colour having a light absorption peak which varies in wavelength according to the precise point at which hydrolysis is terminated. In other words, the purple colours are mixtures of varying composition made up of the blue (from DP > 40 zone) and the red (from DP 25 to 40 zone) colours and the linear relationship is no longer maintained when hydrolysis has proceeded as far as the purple colour-stage. During preliminary experiments on a series of dilutions of serum of low amylase activity and with amylose as substrate, it was observed that on addition of iodine some of the mixtures gave a purple colour which faded rapidly to a turbid yellow colour. This fading was subsequently attributed to the presence of an excess of serum protein, which presumably competes with the carbohydrates in the hydrolysate for the iodine. KeflWncesp. 268

258

H. V. STREET,

A method

using amylose

amylose-iodine-iodide

complex

as substrate to measure

VOI.. 1

1. It.C‘LOSE:

and employing the amount

the blue colour

of amylose

(I()jO)

of the

and blue-staining

dextrins remaining after termination of the hydrolysis was therefore considered worthy of investigation. An account of these investigations is presented here together with a proposed method for determination The

method

IOO patients activity.

of the activity

of amylase

has also been used to determine

in serum and in urine.

the serum

in whom there was no reason to suspect

amylase

any abnormality

activity

of

of amvlase

ESPERIMENT.ll. In all experiments, unless otherwise stated, all the calorimetry was carried out in a Gambrel1 single cell photoelectric calorimeter fitted with a scalp divided logarithmically. A red (Ilford No. 204) filter was used and the zero of the instrument with distilled water. Preparation

of amylose

SCHOCH 21and HOHWN from starch.

was set

et al. 22 have reviewed methods for preparation

of amylose

We have used a method similar to that of H.&WORTH et al. 25for isolation

of the amylose. It is simple to perform, very pure with respect to contamination 30 g of starch (R.D.H.)

does not employ dialysis and the product by amylopectin.

is

were made into a cream by stirring with 150 ml of distilled

water. The cream was then poured into Ijoo ml of boiling zo/1 (w/v) sodium chloride solution. The hot mixture was stirred mechanically and vigorously until it was homogeneous and then filtered, whilst hot, through muslin. To the cooled filtrate were added 4.5 g of thymol

(powdered crystals)

for 48 hours. The supernatant

liquid,

The precipitate

washed six times ethyl

alcohol.

containing

of the amylose-thymol with water saturated

The precipitate

and the mixture amylopectin, complex

was decanted

was removed

with thymol

was centrifuged

was stirred mechanically and discarded.

by centrifuging

and

and four times with absolute

down in between

each washing

and,

after the last washing, was spread out on a glass plate and dried in a 37 “C incubator for six hours with occasional grinding in a mortar. This process causes the amylose to retrograde, i.?., it becomes almost insoluble in water. However, this is of no import for our purposes as WCwere able to disperse the amylose (using ethanol as a wetting agent) in hot dilute alkali to give a clear, colourless, stable solution. The yield of amylose was usually about 3.0 g. The white powder was finally transferred to and stored in an amber-coloured glass reagent bottle with a tightly-fitting stopper, and kept at room temperature. This amount of amylose is sufficient for over 500 determinations of amylase activity when performed singly, i.e., including two dilutions of sample and one standard for each determination.

During the preparation of solid amylose, it is essential to carry out the operations following the decantation of the amylopectin as quickly as possible and to dry the product in a vacuum if a water-soluble product is to be obtained. We have found it more satisfactory to carr!. out the operations at normal speed, to allow the amylosc K~~/~~VerKL~s p. 2hh

VOL. 1 (1956)

AMYLASEACTIVITY IN BIOLOGICALFLUIDS

259

to retrograde and then take up the solid in alkali. Amylose solutions in alkali do not retrograde. Various solvents were tried initially, e.g., boiling water, hot saturated aqueous potassium iodide, hot saturated sodium chloride, but the one which seemed most satisfactory was 0.01 N NaOH solution. IOO mg of amylose were weighed in a 20 ml beaker and wetted by addition of about 2 ml of ethanol. The mixture was stirred with a fine glass rod and poured into a 250 ml beaker containing about 80 ml of 0.01 N NaOH solution which had previously been heated to about 90 “C just before it was required (and the flame then removed). The smaller beaker and the stirring rod were rinsed with further portions of ethanol until complete transfer of the amylose had been effected. Not more than a total of 5 ml of ethanol were used for dispersion and rinsing. Most of the ethanol boils off as it reaches the surface of the hot alkali. That which remains does not interfere with the determination. The mixture was cooled by carefully holding the beaker under cold running tap-water, and transferred to a IOO ml volumetric flask. The beaker was rinsed out several times with small volumes of 0.01 N NaOH solution and the washings added to the IOO ml volumetric flask, the contents of which were finally diluted to IOO ml with 0.01 N NaOH solution and well mixed. If this solution is exposed to the carbon dioxide of the air for 12 hours, the amylose starts to precipitate. It is recommended, therefore, that the solution be stored in a stoppered polyethylene bottle. Criterion

of purity

of amylose

Most of the workers in this field of carbohydrate chemistry have taken the “blue value” described by HASSID AND MCCREADY24as the criterion of purity of the separated components of starch. This blue value is the intensity of the blue colour of the amylose-iodine complex prepared under standard conditions and measured at a particular light wavelength in a suitable calorimeter. As not all clinical laboratories possess the same type of calorimeter, it seemed to us to be of little use to quote numerical values for this “blue number” as a guide for other workers as to the purity of their separated product. Instead, we have taken as the criterion of purity of our amylose preparations, the fact that no amylopectin can be demonstrated when the preparation is subjected to (I) electro-osmosis (GREENWAY et al. 25-hut see also a criticism of this by MOULD AND SYNGE 19)and (2) the leaching action of a particular solution. This leaching action was carried out in exactly the same manner as for descending paper-chromatography (see CONSDEN et al. 26) to which, incidentally, it bears some resemblance. 2.5 ~1 of 1.0% solutions of amylose and of starch in 0.2 N NaOH were placed as “spots” about 3 cm apart on a strip of Whatman No. I filter paper. When the “spots” had dried, the paper was hung from a trough inside a chromatography tank. Borate buffer, pH 8.0, was placed in the trough and was allowed to flow down the paper and to drip off the end of the paper. This irrigation was allowed to proceed at room temperature for 65 hours, during which period the tank was sealed by a glass lid. At the end of this time, the strip was removed and dried in a current of cold air. It was then immersed in 0.02 N iodine solution for about 15 seconds and then rinsed with water at about 45 “C for approximately 5 seconds. This rinsing with warm water removes the background stain on the paper but does not greatly affect the intensity Keferencesp. 1&s

Starch

Amyfo.%e

Fig. t. Eflect of leaching action on amylose md starch. Borate buffer pH 8.0, 0.5 hours. 50 ml 0.2 ~11 H,RO, in 0.2 J1 KC1 + 3.~~7ml 0.1 N NaOH diluted to 100 1711.

starch

solutions.

starch

shows a similar blue streak about 25 mm in length and, in addition, a reddish-

The amylosc

appears

as a hluc streak

about 40 mm in length;

the

coloured streak about IIO mm in length. The red streak is due to the amylopectin in the starch. It will be noticed that the amylose “run” shows no trace of red-staining amylopectin, and also that the solutions used were ten times the concentration finally adopted for the estimation. We feel, therefore, quite justified in accepting the results illustrated

by Fig.

I

as an indication

of an amylopectin

low to be negligible for our purposes. The results of clectro-osmosis are similar

to the

content

at least sufficiently

“pseudo”

chromatography

described above. Whilst, however, no amylopectin could be demonstrated in any of our amylose preparations, the resolution, by electro-osmosis, of the two starch components

was not as good, nor the results as reproducible

as with the “leaching”

procedure. Vnria&ion of optical Izepzsit?iof &te amylose-1,-I’

complex with ~rn~~~~l~ ofiodine added

z ml of o.:y:;, amylose in 0.01 K NaOH were pipetted into each of a series of IOO ml volumetric flasks containing 5 ml of 0.02 M phosphate buffer (pH 7.0) and 50 ml of distilled water. z ml of 0.01 ,V HC1 were added followed by different volumes of 0.01 N iodine in 0.3% (w/v) KI. The contents of each flask were mixed and then diluted to roe ml with distilled water. After thorough shaking, the solutions were compared colorimetricnlly. Table I illustrates the results obtained. Identical readings were obtained when 5 ml of ethanol were added to the flasks prior to addition of iodine. These results show that, under the conditions prescribed, 4 ml is a suitable volume of the iodine solution to give maximum intensity of blue colour and that small amounts of ethanol remaining in the amylose solution have no Referencesp.

rbS

VOL,

1 (1956)

effectat formed

AMYLASEACTIVITYINBIOLOGICALFLUIDS

261

least an the intensity of the blue colour. These experiments with and without a blank, prepared as above but omitting

EFFECT

OF DIFFERENT

AMOUNTS

OF

1,

O;“a XSTENSITY

OF BLUE

were also peramylose. The

COLOUK

differences in the readings were negligible. It was, therefore, decided to omit an iodine-blank in the final method. The actual concentrations in the Bask c~~t~~~i~g 4 mI of iodine solution were: iodine, 4 * m-4 &f; potassium iodide, 7.5 . TO-$M; amylose, 2 mg per IOO ml (CA MoULD~').

The absorption curve (from 400 to 700 rnp) of the amylose-iodine complex has been measured od a UNICAM SP, 500 Quartz Spectrophotometer and is shown in

I

I

au0

500

I

I

go0

700

Wavelengthbry) Fig. 2.

Absorption curve of amylose-iodine Jlfnrd

Filter

complex superimposed No. 204 (see text).

on the transmission

curve

of

Fig. 2. This solution was prepared by mixing 2 mlofo.~Si, amylose in o.or N WaOH solution, z ml of o&or N HCl, 5 ml of 0.02 M phospl~atc buffer (p&I 7.0)‘ 4 ml of O.QI N iodine in O.J% KI and diluting to roe ml with distilled water, (i.e. the same solution as used for “standard” in recommended procedure). The transmission curve of the llford No. 204 filter has been superimposed upon the amylose absorption cur-w. It will be observed that the filter is suitable for measurement of the optical density of the blue solution. K&W?I&L’S i”t.268

Into each of nine zoo ml volumetric flasks were placed j ml of 0.02 22 l~hosl~hatt~ buffer (pH 7.0), 2 ml of 0.01 ,V HCl, x m1 of o.IS;;~ amylose in 0.0~ .\’ NaOH solution and 50 ml of distilled water. The amou~~tof 0.01 X NaOH was maintained the same in each flask, (G., 2 ml), P.E., when 0.7 j ml of amylose solution was ackkd, I .25 d rJf 0.01 iv NaOH were added. 4 ml of 0.01: S I, in 0.3?~:, KI solution were then added and the contents diluted to IOO ml with distilled water. After tlloro~lg~~shaking and mixing, the blue solutions were compared calorimetrically. The results arc illnstratt~d graphically in Fig. 3, which shows that there is a linear relationship between colorimeter readings and amount of amylose added. In order to examine the stability of the blue colour, the solutions were read again

after being allowed to stand for 15 minutes. Identical readings were obtained. Furthermore, addition of an equal volume of distilled water to the blue solutions (at room temperatures gave readings (log scale) which were exactly one half of what they were before dilution. The results show that the blue amylose -I,-I’complex obeys Beer’s Law. We have also demonstrated the fact that identical readings are obtained wheu the flasks are placed in a 37 “C water-bath for 15 minutes prior to addition of the 50 ml of distilled water. Relationship

betweeu chloride ion corccentration and umyluse activity

In these experiments, a number of IOOml volumetric flasks were used, each containing 5 ml of 0.02 &I phosphate buffer (pH 7.0), 2 ml of 0.17~ amylose in 0.01 .?d XaOW solution and 2 ml of 0.01 N I-ICI solution. 0.4 ml of serum was diluted with x.6 ml of x IL’ NaCl solution and z mi of this mixture was added to and incubated at 37 “C for 15 minutes with the contents of the IOO ml flasks. Cold distilled water was then added, followed by 4 ml of 0.01 N iodine in o,3y0 KI solution, The mixture was diluted with distilled water to IOO ml and the blue colours compared calorimetrically. The normality (x) of N&l solution was chosen so that the chloride ion concentration in the incubation mixture ranged from 4.0 . 10-3 to 1.04 * IO.-'N. The results are expressed graFhi~lly in Fig. 4. The first portion of the curve shows that as Cl‘ concentration increases so does amylase activity until the Cl References Q. 268

VOL.

1 (1956)

AMYLASE

ACTIVITY

IN BIOLOGICAL

263

FLUIDS

concentration reaches about 0.01 N. From this point onwards, up to at least 0.1 N, increasing Cl- concentration has no further effect on amylase activity. In view of these results, therefore, 0.1 N NaCl solution was chosen as a suitable

g::l, , , , , , , , , , , g

00

10

20

Cl’

e 3

30 40 50 60 70 00 90 100 no concentration in incubation mixture(N

Fig. 4, Effect of increasing Cl- concentration

and convenient this purpose. Relationship I,-I’-comfilex

diluent

x1000)

on amylase activity.

for the serum and has been used throughout

between degree of dilution of serum and optical

this work for

density of the amylose-

In these experiments, a standard was used containing a known weight of amylose. A number of different dilutions of a sample of serum was made. These diluted sera were incubated with the same amount of amylose as that contained in the standard. The difference between the optical density of the blue colour produced by the standard and that produced by a diluted serum was taken as a measure of the amylase activity of that particular dilution of serum. Into each of nine IOO ml volumetric flasks were measured 5 ml of 0.02 M phosphate buffer (pH 7.0), 2 ml of 0.1% amylose in 0.01 N NaOH solution and 2 ml of 0.01 N HCl. Eight of these flasks were placed in a 37 “C water-bath for 3 minutes to allow the contents to assume the temperature of the bath before the sera were added. Dilutions of serum were then made as shown in Table II. TABLE11 ml of 0.1 N NaCl

I.0

I.4

I.5

1.6

I.7

0

I.0

I.5

ml of serum

1.0

0.6

0.5

0.4

0.3

0

0

0

0

0

0

0

0

2.0

1.0

0.5

50

30

“5

5

2.5

ml of serum diluted

I in

IO with 0.1 N NaCl o/o neat serum mixture (x)

in final 20

15

10

To the ninth flask, which was marked “Standard”, was added 2.0 ml of 0.1 N NaCl. 1.0 ml of the diluted serum was placed in the eight flasks in the bathandincubation was allowed to proceed for exactly 15 minutes. At the end of this time, the flasks were removed from the bath and the contents were immediately diluted with cold (ca. IO “C) distilled water to about 80 ml. The contents of the “Standard” flask were also similarly diluted. 4 ml of 0.01 N iodine in 0.3% KI solution were then added References

p.

268

to cxach flask and the contents diluted to roe ml with distilled water. LAfttr thorouglr shaking and mixing, thr. solutions were compared colorimctricall!,. Thr~ reading givc)n hy each diluted serum (7’) and the reading of the standard (S) were incorporated in tfre formula : hmylase

activity

= ‘.F (S--T)

units per I00 ml of serum.

Hence we can relate the amylase activity to the amount of amylosc which has undergone hydrolysis. If we assume that as soon as all the amylosc has been hvdrolysed, the mixture gives no colour on addition of iodine, then amylase

activity

-= ‘y

(S-o)

units per 100 ml of serum.

= 100 units per I00 ml of serum. Now 2 mg of amylose are contained in the digestion mixture to whichthe equivalent of 0.1 ml of neat serum is added. Our units, therefore, may be defined as follows: a fluid has an amylase activity of IOO units per IOO ml when 0.1 ml of the fluid will cause the complete hydrolysis of z mg of amylose (under the conditions prescribed in our method) at a pH of 7.0, a temperature of 37 “C and for an incubation period of 15 minutes. Fig. 5 shows a typical curve obtained by plotting amylase activity against amount

75

0

70 65

% Neat

serum

in

fmal

dilution

(x)

of serum in the digest. This experiment was performed several times using serum of low, normal and high amylase activit!y. In all cases, a linear relationship was

VOL. 1

(1956)

AMYLASEACTIVITYINBIOLOGICALFLUIDS

2%

obtained over the range of activity o to 60 units. Above this value, the curve is not linear, and the colours produced which give rise to these values are not blue, but may vary from mauve through red to yellow (see MOULD AND SYNGE 18). During the course of these experiments, it was observed that, when relatively large volumes of serum were added to the digestion mixture, the iodine colours produced were atypical. This was shown to be due to combination of the iodine with the serum proteins, leaving insufficient iodine for combination with the polysaccharides. Under the conditions described in our “Final Method”, this phenomenon does not occur.

METHOD

FINALLY

ADOPTED

Reagents

It is recommended that all reagent solutions, except the iodine solutions, be stored in bottles made of polyethylene. 0.1 o/o Amylose solution. IOO mg of amylose (prepared as described above) are weighed in a 30 ml beaker. Small volumes of 99 to 100% ethyl alcohol are used to transfer quantitatively the amylose from the 30 ml beaker to a 150 ml beaker containing about 70 ml of 0.01 N sodium hydroxide solution maintained at a temperature of approximately 85 “C. Not more than 5 ml of ethyl alcohol should be used to effect this transfer. The contents of the 150 ml beaker are allowed to cool to room temperature and then poured into a IOO ml volumetric flask. 0.1 N sodium hydroxide solution is used to rinse out this beaker and to dilute the amylose solution to TOOml. 0.02 M Phosphate bufler solution, pH 7.0. 3.471 g of anhydrous disodium hydrogen phosphate (Na,HPO,) and 2.118 g of potassium dihydrogen phosphate (KH,PO,) are dissolved in distilled water and the solution is diluted to 2 litres with distilled water. N Hydrochloric acid solution. Stock iodine solution (0.1 N). 30 g of potassium

0.01

iodide are dissolved in about 250 ml of distilled water. 13 g of iodine are dissolved in this solution, which is then diluted to I litre with distilled water. 0.01 N Iodine solution. IO ml of the stock (0.1 N) iodine solution are diluted to IOO ml with distilled water. 0.1

N Sodium chloride solution.

Serum dilutions. (a) I in

IO diluted serum. 0.2 ml of serum is thoroughly mixed with 1.8 ml of 0.1 N sodium chloride solution. (b) I in 40 diluted sewm. 0.2 ml of serum is thoroughly mixed with 7.8 ml of 6.1 N sodium chloride solution. Urilze. By appropriate dilution, the method is also applicable without modification to urine.

Suggested procedure

Into each of three IOO ml volumetric flasks, marked “Standard”, “Test IO" and “Test 40”, are measured 5 ml of phosphate buffer, 2 ml of 0.1% amylose solution and Referencesp.

268

2 ml of 0.01 .\’ hydrochloric acid solution. ‘l‘hc flasks marked “Test IO” and “Test 40” are placed in a 1)cakcr of water in a water-bath at .37 “(‘ for .: tnitlutc‘s to allow thts contents of the flasks to assume the temperature of the bath. One ml of I in TO diluted serum is plactd in “Test 10” flask and I ml of r in 40 diluted serum is placed in “Test 40” flask. The flasks are thcu allowed to remain in the bath for exuctl? 15 minutes after the addition of the dilntcd scra. At the end of this time, the flasks are removed from the bath and the contents are immediately diluted to approximately So ml with distilled water. ~\hout the same volume of distilled water is also added to the flask tnarked “Standard”. ~~itl~o~lt delay after the addition of the water, 4 ml of o.or S iodine solution are added to all three flasks. The contents of each flask are diluted to 100 ml with distilled water and mixed. The colours are compared in ;I photoelectric calorimeter using a red filter or in a spectrophotometer at 020 m!u. IMilled water is used to set the zero of the calorimeter.

The I in IO dilution of the serum covers the range of amylase activity from zero up to about 30 of our units per 100 ml. If the colour of the contents of the flask containing the I in IO dilution of serum is not blue-green but has a reddish or mauve tinge or is yellow, then the result is calculated from the reading of I in 40 diluted serum. If the I in 40 dilution of serum does not show a blue-green colour, then the procedure must be repeated using a greater dilution of serum, say I in 75 or I in loo. I;O?’

I

in IO

dihtiOF1

Serum amylasc

activity

IhF(S--Y‘)

units per I00 ml.

4g” (5-7’)

units per I00 ml.

For I i~z$0 ~~~~~~{)~~ Serum amylase

References

P. 26X

activity

VOL. 1 (1956)

AMYLASEACTIVITYIN BIOLOGICALFLUIDS

267

APPLICATION In order to gain some idea of the range of our units in “normal” persons we determined the serum amylase activity of IOI hospital patients (both in- and outpatients) in whom there was no reason to suspect an abnormal serum amylase activity. The results of these investigations are illustrated graphically in Fig. 6. The arithmetical mean is 19.08 units and the results range from 6 units to 40 units. g y0 of the results are less than IO units and 8 o/o of the results are greater than 30 units: 2 o/o are less than g units and 2 per cent are greater than 35 units. Until further results on “normal” healthy persons are available we have adopted the range of g to 35 units as a temporary working “normal” range. ACKNOWLEDGEMENTS We wish to thank Dr. J. DAVSON for his helpful criticism of the manuscript and for valuable suggestions. We are grateful to Mr. R. HORROCKS, B.Sc., F.R.I.C., for preparation of the absorption curve of the amylose-iodine complex (Fig. 2). Our thanks are also due to Miss JEAN PERRY for preparation of the figures and to Miss H. BURLEY for secretarial assistance. SUMMARY A rapid method for the calorimetric determination of amylase activity in serum and urine is described. This method uses amylose as substrate; it is very simple to perform, uses only 0.4 ml of serum, and can be completed in approximately 25 minutes. Preparation of amylose and a criterion of purity of the product are given. The influence of various pertinent factors has been examined and is discussed. Application of the method to LOOcases and a working “normal” range are given. RESUME Les auteurs decrivent une methode de determination colorimetrique rapide de l’activite amylase dans le serum et l’urine. Dans cette methode, l’amylose sert de substrat ; il ne faut que 0.4 ml de serum; la determination est tres facile a executer et peut &tre terminde en 25 minutes environ. La preparation de l’amylose et un critere de pureti: du produit sont decrits. Les auteurs ont examine et ils discutent l’influence de divers facteurs pertinents. 11s decrivent l’application de la methode a IOO personnes chez qui l’on n’avait aucune raison de s’attendre a une activite amylase anormale et indiquent un domaine de valeurs considere temporairement comme “normal”. ZUSAMMENFASSUNG Eine rasche kolorimetrische Methode zur Bestimmung der Amylaseaktivitat im Serum und Urin wir beschrieben. Amylose dient als Substrat; man benijtigt nur 0.4 ml an Serum; die Bestimmung ist sehr einfach auszuftihren und kann in ungefahr 25 Minuten beendet sein. Die Herstellung der Amylose und ein Kriterium fur die Reinheit des Produktes

VOI.. 1 (r()p)

H. V. STHE:ET, J. Ii. C’LOSI-

ZOS

werden beschrieben. Der EinfluW einer Anzahl Faktoren wurde untersucht und wird eriirtert. Die Methode wurde auf IOO Personen angewendet, hei denen keinc abnormale Amylaseaktivitat zu erwarten war und es wurde ein Bereich von Werten angegeben, der vorlaufig als “normal” betrachtet werden kann. PE3K)ME AaHo 0nxcaHIle ~~ICT~OI-0MeToAa KonopriMeTprnecrioro onpe~eneakra aKTnr3nocni aMkfna3br B cepyMe II M09e. npa EJTOM aMEmo3a CAYXKEIT B KaqeCTBe cy6cTpaTa. Meron 3~0~ OgeHb npocT M Tpe6yeT TOA~KO 0,4 MA. cepybfa II 6epeT npH6nI13uTenbHo 25 MIIHYT. Aaercn cnoco6

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REFERENCES I

2 3 4 5 6 zi 9 IO II 12 13 14 I5 16 I7 18 I9 LO ZI 22 ‘3 24 25 26 27

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Received

April 6th, 1956