An Improved Photom etric Method for Lipase Activity Suitable for Both Routine and Reference W ork*

Transcription

1 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, Vol. 2, No. 6 Copyright 1972, Institute for Clinical Science An Improved Photom etric Method for Lipase Activity Suitable for Both Routine and Reference W ork* CHARLES G. MASSION, M.D. AND MICHAEL D. McNEELY, M.D. University of Connecticut Health Center, McCook Hospital, Hartford, CT ABSTRACT Modifications that optimize the conditions and reduce the complexity of a sensitive and accurate serum lipase assay have been described. The optimum temperature for performance was shown to be 37 C and the proposed substrate emulsion was revealed to be stable for four weeks while lipase activity in pooled and frozen human serum was found to be constant for 11 months. Olive oil was confirmed as an adequate source of triglyceride compared to other commonly available sources. Introduction T he association b etw een increased serum lip ase activity and pancreatitis has b een clinically u seful since th e introduction of a practical analytical m ethod b y Cherry & C randall1 in U nfortunately, this m ethod is encum bered b y the 16 to 24 hour incubation required to release sufficient fa tty acid from th e triglyceride substrate to allow m easurem ent b y titration. M any m odifications h ave b een published in an effort to increase th e sensitivity and reduce th e incubation tim e. W h ile som e o f these reports h ave h elp ed to define the conditions necessary for reproducible results, other procedures ad vocating th e u se * Supported in part by Research Funds (Part II) from the U. S. Veterans Administration. of substrates other than lon g chain triglyceride5 9 h ave produced considerable confusion sin ce hydrolysis of these substrates actually m easures the activity of esterases other than lipase. Pancreatic lip ase is sepecifically defined as differentiated from other esterases in that it only acts on an ester-w ater interface and is relatively ineffectual against w ater soluble substrate. 4 This definition excludes the w ater solu ble esters and short-chain triglycerides that h ave b een u sed in som e m ethods. Som e o f the m ethods used h ave not b een properly designated as esterase assays, b ut h ave b een m istakenly called lipase procedures. In 1967, M assion and Seligson described a rapid photom etric m ethod for the precise m easurem ent o f lipase activity.7 This m ethod u sed an olive oil substrate and related enzym e activity to purified fatty acid standards. This techn iqu e of m easurem ent of activity has b een u sed for several years and th e assay has b een further d e velop ed b y em ploying a standard curve that is m ore u seful than the previous on e.t In addition, th e optim um tem perature for incubation has b een defined, th e stability of th e recom m ended substrate em ulsion has b een established, th e p ossib le advantages o f triglyceride substrate sources other than 4 4 4

2 IMPROVED PHOTOMETRIC LIPASE METHOD 445 olive oil h ave b een investigated and th e inherent variability of th e blank and test has b een exam ined. T h ese findings and a revised m ethod for lipase activity determ ination w ill b e presented. Principle A buffered em ulsion of liquid triglyceride w as hydrolyzed b y serum lipase so that lon g chain fatty acids w ere released. T h e acids and excess oil w ere extracted into a fat solvent, w h ich was then evaporated. T h e fatty acid-in-oil residue w as m ixed w ith a buffered indicator solution that changed color as a function of the fatty acid content. R eagents'and Solutions R eagents Absolute ethanol, reagent grade. Sulfuric acid, 0.28 N. Four m l o f concentrated sulfuric acid are ad ded to 500 m l of water. Acid-alcohol mixture. Three volum es of absolute ethanol are added to tw o volum es of 0.28 N H 2SO4. This solution m ust b e prepared fresh daily. Petroleum ether. (B.P. 30 to 6 0 C ), redistilled. Ethanol 95 percent, reagent grade. Methyl red 0.2 percent. (F ish er Scientific Co., M -219). T w o hundred m g of pow dered m ethyl red w as dissolved in 100 m l of 95 percent ethanol. Solution w as slow, requiring 48 hours. This can b e reduced to 12 hours by use of a m agn etic stirrer. F in al concentration w as adjusted photom etrically. Olive Oil, reagent grade (F ish er Scientific Co., No ). O live oil w as readily and m ost com pletely freed from fatty acids by passing through alum ina (M erck, N o ) contained in a colum n or separatory funnel p lugged w ith glass w ool. O nly one volum e of oil can b e adequately purified by an equal volum e of alum ina. T he fatty acid content (blank valu e) o f th e oil w as checked b y p ipettin g a m l aliquot into 3 m l of m ethyl red reagent, and then shaking and centrifuging th e m ixture leavin g th e fatty acids in solution. If th e absorbance increased m ore than in 10 m m cuvettes, th e o il w as passed through m ore alum ina. If refrigerated at 4 C, th e oil retained a low blank value for at least one w eek. Com Oil. Grocery item, purified sam e as olive oil. Cottonseed Oil. Grocery item, purified sam e as olive oil. Triolein, Chromatographic Grade. (M ann R es. Lab ). U sed as received. Substrate/Buffer, Triglyceride Emulsion 5 percent. Purified oil, 5 m l, w as h om ogen ized in a high-speed household blender w ith 95 m l of buffer solution. R ep eated short periods of blen ding (3 0 to 60 secon ds) w ere follow ed b y short intervals of cooling to avoid over-heating of th e b ushing in the blender base. H eating appeared to cau se significant hydrolysis o f th e triglycerides in th e oil. If blen ding w as p erform ed in a single continuous operation, th e base becam e very warm and th e blank valu e o f th e b atch w as substantially elevated. B lending w as continued until n o film of oil w as present after th e foam settled. T he p H w as adjusted to 8.50 at 25 C b y th e addition o f IN HC1, or N ao H. T he em ulsion w as stored at 4 to 6 C. A 100 percent elevation of blank value w as u sed as an indication for preparing a fresh su bstrate/buffer reagent. Buffer. A liter o f buffer w as prepared b y sequentially com bining 2.42 g Tris ( hydroxym eth yl) am inom ethane, 3.5 g deoxycholic acid and 0.2 g sorbic acid in a one liter volum etric flask and diluting to the mark w ith distilled w ater after adequate m ixing. Methyl red reagent ( buffered indicator solution). This reagent w as prepared b y photom etric m easurem ent to avoid variations in d ye lots. T en m l of 1 N N ao H w ere ad ded to 1 liter o f 95 percent ethanol. E nough 0.2 percent m ethyl red solution

3 446 MASSION AND MCNEELY ( usually 10 to 13 m l) w as ad ded to bring the absorbance to to A in 10 m m cuvettes using a w ave length of 500 nm. O ne m l of 1 M sodium acetate w as added, using a m agnetic stirrer for m ixing, and 1 N hydrochloric acid w as added dropw ise until a faint orange-red tinge persisted. M ore acid w as added carefully until the solution had an absorbance of ± in 10 m m cuvettes. If this point was passed, the solution w as back titrated w ith dilu te N ao H. E xcessive dilution of these aqueous solutions should b e avoided. T h e color of the reagent m ay fade slightly in th e first few hours, b u t thereafter w ill rem ain unchanged for at least a m onth if it is stored at room tem perature in dark brow n glass bottles. E ventually, how ever, th e color w ill change. T he use of bleached solutions is not recom m ended. Standard So lutio n Stearic Acid (F ish er Scientific Co., No. A -293). A solution containing 1.0 /xeq per m l w as prepared b y dissolving 28.5 m g o f stearic acid in 100 m l of heptane or petroleum ether. T he form er w as preferred b e cause of th e higher boiling point. T he standard curve w as set up to read directly in m icroequivalents of fatty acid produced. C arefully m easured portions o f standard solution equivalent to 0.25, 0.50 and 0.75 /xeq (0.25, 0.50 and 0.75 m l) w ere placed in screw -topped tubes and w ere dried in a 50 to 60 C w ater bath w ith a gen tle stream of air to hasten drying. Several sets of standards can b e prepared at once since the dry residue is stable for w eeks in tightly capped tubes. Special Apparatus A device to accelerate the evaporation of petroleum ether from the fatty acidolive oil m ixture b y delivering a filtered stream of air into each tube w as extrem ely h elpful and reduced th e perform ance tim e b y 10 to 15 m inutes. Such a device w as easily constructed by cem enting 4 inch lengths of thin m etal or rigid plastic tubing into holes drilled in th e bottom of a sm all w ooden or plastic box. The box w as then filled w ith loosely pack ed glass w ool. C om pressed air was injected into one side of th e box and distributed evenly b y the lengths of tubing into the tubes containing petroleum ether-fatty acid solution. E vap oration w as further hastened by placing the tubes in a 50 to 60 C w ater bath or dry block. Method M easurem ent of F atty A cids Released A liquots o f serum and of substrate w ere prew arm ed for five m inutes in a 37 C w ater bath. O ne m l of substrate and 50,«1 of serum w ere p ipetted into a screw-top glass culture tu b e and w ere m ixed. The tubes w ere cap p ed or covered. T he m ixture w as incubated for exactly 30 m inutes. Serum and substrate w ere in cub ated sim ultaneously and separately also. A cid-alcohol (3.3 m l) was added to stop the reaction and to convert th e form ed fatty acid salts into the acid form. Acidalcohol was also added to 1 m l of substrate in a separate tube and 50 A o f serum was then added to produce a sam ple blank. A reagent blank w as also prepared by com bining 3.3 m l acid-alcohol and 1 m l of substrate mixture. T he reagent blank was not used except to check stability of the em ulsion. Four m l of petroleum ether w ere added to all tubes. T he sam ples w ere tightly capped and w ere shaken vigorously b y hand for two m inutes and then centrifuged at 2,000 rpm for five m inutes. A 2.0 m l aliquot of the petroleum ether phase (to p layer) w as rem oved and placed in another glass screw-top tube. D uring this step, great care w as taken to avoid the interface w ith th e tip o f th e pipette since significant am ounts of m ineral acid are

4 IMPROVED PHOTOMETRIC LIPASE METHOD 447 easily picked up. The petroleum ether was evaporated in a 50 to 60 C water bath with a gentle stream of air. Three ml of methyl red reagent were added to the oil residue and the tubes were capped with teflon-lined caps and shaken vigorously for one minute. The tubes were centrifuged at 2,000 rpm to remove the suspended oil. An angle head will not allow good removal of the olive oil. The supernatant was decanted from the drop of olive oil and read in 10 mm cuvettes in a spectrophotometer with the wavelength set at 502 nm. Samples exhibiting an absorbance greater than the highest standard should be diluted with an equal volume of methyl red reagent. This dilution may be repeated if necessary. If dilution is necessary, the serum blank must be diluted equally. Three ml of methyl red reagent were added to each of the three tubes in a set of standards (0.25, 0.50 and 0.75 / Eq). The tubes were then tightly capped and shaken until the stearic acid was completely dissolved. The zero was set with alcohol as a bl anv Absorbance v s I u g s of tli0 mctlivl red reagent blank and of the standards were measured and plotted on linear graph paper. C alculations The amount of fatty acid in the samples and blanks was determined by referring the measured absorbances to the standardization curve or by calculation of the line slope or k factor and then applying the formula: (/ Eq sample / Eq serum blank) ( dilution) ( total extraction volume /volume used) (1) / (serum volume) = / Eq fatty acid released per ml of serum. More simply: (net fatty acid released) X (dilution X 2 X 20 = units of lipase where 1 /teq = 1 unit of lipase activity/ml of serum. Norm al Range Units per ml of serum. Experim ental S t a n d a r d C u r v e s C o m p a r i s o n Two batches of methyl red reagent were prepared. These were identical except that one contained two mmol per liter sodium acetate and the other one mmol per liter. Two sets of standards were prepared containing amounts of stearic acid from 0.25 to 1.50 /teq. Two standard curves were then developed by adding three ml aliquots of one batch of methyl red to one set of standards and three ml aliquots of the other batch of methyl red to a duplicate set of standards. After the dried standards had dissolved in the color reagent, the absorbance was read on a precision spectrophotometer. O p t i m u m T e m p e r a t u r e f o r I n c u b a t i o n Three pools of human serum were prepared, one each with normal, slightly increased and greatly increased lipase activity. The activity of each pool was determined with incubation at 30, 35, 37, 40, 42.5 and 45 C. S u b s t r a t e S t a b i l i t y A batch of olive oil substrate was prepared and utilized for a set of measurements and then refrigerated. Each week for five weeks an aliquot of the emulsion was warmed to 37 C in a water bath and shaken by hand for about one minute. This portion of previously prepared substrate was then compared with a freshly prepared olive oil emulsion by performing replicate lipase measurements on each, using pooled normal and active serums. E n z y m e S t a b i l i t y A large pool of moderately active serum was prepared by diluting a pool of five active serums with human serums of normal activity. This pool was then divided into one ml aliquots, was frozen and was stored at 20 C. Over a 12 month period, the activity of the pool was measured twice

5 448 MASSION AND MCNEELY fatty acid concentration did not produce an exactly straight line, although portions of the curves closely approached linearity as is demonstrated in figure 1. F ig u r e 1. Comparison of standard curves obtained by different concentrations of sodium acetate buffer. The maximum deviation from linearity (as shown by the dashed line) between the 0.25 and 0.75 / Eq is 0.5 percent at the midpoint. each week on a freshly thawed aliquot using a freshly prepared substrate emulsion. T r i g l y c e r i d e S o u r c e Four different substrate emulsions were prepared using olive oil, com oil, cottonseed oil (all purified as previously directed) and triolein as purified by the manufacturer. Aliquots of each batch were incubated with each of 10 serums, both normal and active, and measurements were made with the above method. V a r i a b i l i t y o f T e s t a n d B l a n k V a l u e s Twenty replicate test and blank measurements were made on a normal serum, on a "borderline active serum and on a very active serum, using freshly prepared olive oil emulsion. R e s u l t s S t a n d a r d C u r v e C o m p a r i s o n The standard curves obtained from the two batches of color reagent containing 1 and 2 mmol per liter of sodium acetate buffer are shown in figure 1. The solution buffered by one mmol per liter showed about 40 percent greater sensitivity than the more heavily buffered reagent. The absorbance of the solutions plotted against O p t i m u m T e m p e r a t u r e o f I n c u b a t i o n The relative activity of three pools of human serum at temperatures between 30 and 45 C is shown in figure 2. All the serum pools were relatively less active below 37 C than at that temperature. At 30 C, the activity was reduced about 30 percent. The normal serum remained as active at 40 and 42.5 C as at 37 C, whereas the activity of the two other pools decreased by 12 to 20 percent at the higher temperatures. Above 42.5 C, the lipase activity in all three pools was decreased by 30 to 60 percent. S u b s t r a t e S t a b i l i t y Over a period of four weeks, the average value of triplicate substrate blanks rose from 0.12 /teq to 0.19 / Eq per ml of substrate. For three weeks, the test values of the serum pool rose at the same rate so that the net yield of fatty acid was unchanged. In the fourth week, the net fatty acid yield decreased by 4 percent; by the fifth week, it had declined an additional 17 percent. E n z y m e S t a b i l i t y Duplicate measurements of lipase activity were performed on aliquots of a frozen human serum pool. During the first month following preparation of the pool the activity averaged 21.9 U per ml. For the next 10 months, the mean value of duplicates had a relative standard deviation of 4.6 percent. However, in the twelfth month there was an abrupt decrease of activity to 18.6 U. Further measurements were not made. During the year, the average blank value of the pool (representing hydrolysis of the serum lipids) rose about 10 percent from 0.18 /teq to 0.20 / Eq per ml.

6 IMPROVED PHOTOMETRIC LIPASE METHOD 449 O p t i m a l T r i g l y c e r i d e f o r S u b s t r a t e E m u l s i o n The efficacy of com oil, cottonseed oil and triolein, relative to olive oil, is shown in table I. There was essentially no difference between the yield from olive, com and cottonseed oil, but the yield from triolein was 21 percent lower. The averages of the blank values produced by olive and cottonseed oil were within 3 percent but corn oil had a 14 percent lower average blank value. V a r i a b i l i t y o f T e s t a n d B l a n k V a l u e s Twenty replicates of a serum with normal activity showed a yield of fatty acids with a mean of 0.23 / Eq and a standard deviation of /xeq. Ten blanks yielded a mean of 0.12 /xeq and had a standard deviation of / Eq. Twenty replicates of an active serum had a mean yield of 0.82 with a S.D. of Blanks for this serum had a mean of 0.23 with a S.D. of iteq. The standard deviation of the replicates did not increase proportionately with yield of fatty acids and the relative standard deviation for the active serum was 2.9 percent as compared with 8.7 percent for the normal serum. D is c u s s io n As initially recommended,7 lipase activity was quantitated by comparing the absorbance of the final reactions of unknowns to a symmetrically curvilinear standard curve. The curve was prepared by plotting the absorbance of a blank and five standards containing from 0.5 /teq to 4.0 /j.eq of pure stearic acid. In practice, this span of standards has proven to be unnecessarily large, since even a ten-fold increase in enzyme activity only produced an absorbance change equivalent to the third (2.0 / Eq) in the series of standards. A rare specimen, such as peritoneal fluid, with hugely increased activity (50 to 100 fold) could be read by simply diluting the its TEMPERATURE (*C) F ig u r e 2. The effect of tem perature of incubation on the yield of fatty acids from serums with pooled normal, and moderately active and active serums. Although a least square fit was plotted for each pool, each point is the average of 20 replicate tests and 10 blanks. final solution and blank into the absorbance range spanned by the lower standards. The value obtained will be clinically useful until the incubation can be repeated with reduced sample. It will also give an index of the sample dilution necessary to prevent the substrate concentration from being rate limiting. It became apparent that the complexity of the method could be reduced by utilizing a standard curve that would more closely approach linearity and need fewer than five points to cover a functional range. As can be seen in figure 1, the methyl red reagent containing two mmol per liter of sodium acetate was only slightly curved up to the 0.75 / Eq standard where the absorbance was A. Beyond that TABLE I Y i e l d a n d B l a n k V a l u e s o f T r i g l y c e r i d e E m u l s i o n s R e l a t i v e t o O l i v e O i l Source Yield Blank Olive oil Corn oil Cottonseed oil Triolein

7 450 MASSION AND MCNEELY point, the curve from linearity was more pronounced. By reducing the acetate buffer to one mmol per liter (figure 1), a curve that closely approached linearity and was 40 percent more sensitive was obtained. For purposes of this analysis, the functional portion of the curve lay between the 0.25 jiteq and the 0.75 / Eq standard; it did not include absorbances near the color reagent blank values, since sample blanks had an appreciable absorbance due to the free fatty acid content of both substrate and serum. When linearity was assumed for the functional part of the curve, between 0.25 / Eq and 0.75 /xeq, the maximum error was only +1.3 percent at the midpoint. This represented an error of less than one unit of enzyme activity, an amount too small to produce errors of interpretation. The proposed standard curve spanned up to 22 units of activity or about three times the upper limit of normal. The major advantage of this modification was that absorbance could be transposed into enzyme activity by calculation rather than by comparison with a plotted curve. The optimum incubation temperature for maximum enzyme activity was clearly shown to be about 37 C for serums with increased activity. This is in contrast to the 40 C temperature advocated by Henry.2 While serum containing normal levels of activity did not appear to be affected by 40 and 42.5 C during the 30 minutes incubation time, neither did it increase the yield from enzymatic hydrolysis. Since many enzymes initially show increased activity at increased temperatures, it is quite probable that human serum lipase values obtained after 30 minutes at 40 C reflect that the heat inactivation of the enzyme is greater than the increased activity due to the higher temperature. The possibility that pancreatitis serum lipase is more susceptible to heat denaturation at 40 C than normal serum lipase cannot be ruled out from this data. It is perhaps fortunate that 37 C is indeed the optimum temperature for lipase activity since it obviated the need for an unusual incubator to obtain maximum sensitivity for the assay. This allowed the procedure to be somewhat more readily performed than the earlier version.7 The usefulness of the method was further enhanced by the fact that the olive oil/ buffer emulsion was stable for about a month. It is possible that the stability of the substrate could have been increased beyond a month by sterilization of the ingredients and by keeping the aliquots uncontaminated until used. However, such strenuous efforts seemed unnecessary since the emulsion preparation was neither difficult nor tedious. While serum lipase has been known to be quite stable if refrigerated or frozen,2 3 no information appears to be available on the actual duration of stability of the enzyme in frozen serum. This information is useful to those workers who prepare a frozen serum pool for quality control of lipase measurements. This is frequently necessary since many commercially prepared control materials do not possess lipase activity. The pool utilized in this study contained active serum from six different patients and normal serum from many others and was, therefore, reasonably representative. The precision of activity measurements performed on the frozen pool over eleven months indicated sufficient stability to ensure the usefulness of such pools. Although the decrease in enzyme activity of the pooled active frozen serum in the twelfth month was unheralded by any gradual trend, it did not seem to merit further investigation. Various sources of triglyceride were investigated to find out if any substantial advantage or sensitivity could be gained over the traditional and proven olive oil. The common cooking oils were purified by ex

8 IMPBOVED PHOTOMETRIC LIPASE METHOD 451 posure to alumina. The triolein was not treated since it had been purchased as a highly purified standard. It was unexpected to find the yield of fatty acid from triolein only four-fifths that of olive oil. Since olive oil is composed largely of glycerides of oleic acid,8 this difference may have been due to the removal of an inhibitory substance from the oil by the alumina. The yield of any oil from alumina purification was less than 50 percent. This would have doubled the cost of the triolein and this source was not investigated further because there was no indication of any superiority to offset the increased cost. The comparison of common cooking oils showed com oil to be slightly superior since the average reagent blank was somewhat lower than either olive or cottonseed oil, showing that the emulsion contained the least amount of free fatty acids. This may indicate that the com oil, as purchased, contained less free fatty acids or that the purified oil was less readily hydrolyzed during emulsion preparation. A 14 percent reduction of the blank value of the corn oil did not appear to be great enough to be of any importance since any of the oils could be completely cleared of fatty acid by additional absorption on alumina. However, the acid-free oil was subject to spontaneous hydrolysis, even during refrigeration, and remained relatively clear of free acids for only a few days before gaining an appreciable blank value due to released fatty acids. The most significant cause of triglyceride hydrolysis during the emulsion preparation appeared to be heat from the motor and from the friction of the bushing at the base of the blender. Thus, if the base was allowed to become hot, there was a rise of the blank value of the batch. A certain minimal level of hydrolysis seems to be inescapable unless another method of emulsion preparation, such as sonication, can be utilized. Since some continuing hydrolysis occurred over a period of time in the final emulsion, elaborate methods of purification and preparation to obtain a zero blank seemed unwarranted. However, examination of the variability of replicate measurements of normal and abnormal serums showed cause for some concern because of the relatively wide range of final fatty acid content of both serum blanks and tests. The replicate blanks of normal serum had a mean value of 0.12 microequivalents with a range of 0.06 microequivalents (range = 50 percent of the mean). The test results on normal serum showed a range of 0.08 microequivalents (range = 34 percent of the m ean). Similar results were seen with abnormal serums. Careful pipetting of the volume of substrate emulsion used in each test should have limited the serum blank values to a variation of less than 5 percent. Thus, another fluctuating factor must also have been present to account for the observed range of the replicate assays. There appeared to be a variation of the fatty acid content of aliquots of equal volume of the substrate mixture. One possible explanation is that oil droplets containing dissolved fatty acids were either unevenly distributed in the emulsion or varied enough in size so that a one ml volume of emulsion contained a significantly variable number of oil droplets. Another contributing factor could be the difficulty of repeatedly delivering uniform aliquots of the lipid fluid from non-wetable glassware. The range of fatty acid content of the blank was observed to be only 0.06 microequivalents. Athough this is a small amount, calculation revealed that this range of blank variation was equivalent to 2.4 units of lipase activity. Additionally, the substrate emulsion in the sample tests was subject to the same range of variability (2.4 units). Thus, this range might be doubled to 4.8 units if the lowest possible blank happened to be matched with

9 452 MASSION AND MCNEELY the highest possible test. In actual practice, the calculation of value from 20 replicates of the normal serum showed: Mean test Mean blank = 4.8 U Highest test Lowest blank = 5.6 U Lowest test Highest blank = 1.6 U These worst case extremes were still within the normal range. In fact, they almost spanned the normal range, an observation of some interest, but difficult to interpret. Calculation of values using the replicate results of serum with border line activity showed cause for greater concern. In this particular instance the calculation showed: Mean test Mean blank = 10.0 U Highest test Lowest blank = 14.8 U Lowest test Highest blank = 6.8 U Thus, a marginally active serum could have given both a normal and distinctly abnormal result, producing seriously different interpretations. Superficially considered, this apparently unavoidable sampling error of the acid content of the substrate emulsion droplets would appear to negate any value the test might have for measuring borderline levels of enzyme activity. Statistically considered, however, the worst case of high blank, low test, or the reverse, will occur only once in 1,250 times if a single test is run with a single blank, presuming Gaussian distribution of both measurements. If duplicate tests are run, this will occur once in 72,500 times, hardly often enough to merit further concern in either case. Less marked variation of blank and test results from the mean of replicates will occur more often. Calculation of a borderline active serum, the same example cited above, showed that all combinations of plus or minus two standard deviations from the mean of the test and blank values would produce elevated values that would not lead to misinterpretation. It appears, therefore, that this procedure produced results that are only rarely falsely lowered or falsely elevated when carried out with good laboratory technique. This problem is, of course, not unique to this procedure, but is common to all procedures requiring a sample blank that is subject to variation. The effect of test variability has been frequently considered, but the net result of two variables that occur matched by chance alone has been seldom taken into account. References 1. C h e r r y, I. S. a n d C r a n d a l l, L. A., Jb.: The specificity of pancreatic lipase: its appearance in blood after pancreatic injury. Amer. J. Physiol. 100:266-72, H e n r y, R. J.; Clinical Chemistry: Principles and Technics. Hoeber Medical Division, H arper & How, New York, p. 481, K a c h m a r, J. F.: Fundamentals of Clinical Chemistry, Tietz, N. W., ed. W. B. Saunders, Philadelphia, p. 417, K i n g, J.: Practical Clinical Enzymology. D. van Nostrand Co. Ltd., London, p. 252, K r a m e r, S. P., B a t a t o s, M., K a k p a, J. N., M e n d e l, J. S., C h a n g, A., a n d S e l i g m a n, A. M. : Development of a clinically useful colorimetric method for serum lipase. J. Surg. Res. 4:23-35, M a c D o n a l d, R. P. a n d L e F a v e, R. O.: Serum lipase substrate w ith an olive oil substrate using a three-hour incubation period. Clin. Chem. 8: , M a s s i o n, C. G. a n d S e l i g s o n, D.: Serum lipase: a rapid photometric method. Am. J. Clin. Path. 48: , Merck Index, eighth ed., Stecher, P. G., ed. Merck & Co., Inc., Rahway, NJ, p. 764, P a t t, H. H., K r a m e r, S. P., W o e l, G., Z i e t u n g, D., a n d S e l i g m a n, A. M.: Serum lipase determination in acute pancreatitis. Arch. Surg. 92: , Roe, J. H. a n d B y l e r, R. E.: Serum lipase determination using a one hour period of hydrolysis. Anal. Biochem. 6: , T i e t z, N. W., B o r d e n, T., a n d S t e p l e t o n, J. D.: An improved method for the determination of lipase in serum. Amer. J. Clin. Path. 31: , V o g e l, W. C. a n d Z i e v e, L.: A rapid and sensitive turbidimetric method for serum lipase based on differences of normal and pancreatitis serum. Clin. Chem. 9: , 1963.