The lipolytic activity of the duodenal contents
Craver, Bradford North
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A clinically satisfactory method of lipolytic analysis for the duodenal contents must fulfill eight criteria: (1) and (2) the optimal pH and optimal temperature must be maintained during the course of the reaction; (3) inhibiting agents must be avoided and necessary activators or co-enzymes must be added; (4) operative variables in the system must be held to a minimum by the use of a fixed concentration of a suitable and evenly emulsified substrate; (5) the method of measuring the final extent of digestion must be accurate; (6) the enzyme must be protected from destructive influences in the interim between its procurement and its analysis; (7) the method must be simple, should preferably use ordinary laboratory apparatus and must consume as little time as possible; (8) the method must be designed to give as true a picture as possible of what might be expected to occur in vivo. The following method is presented as more satisfactorily fulfilling these criteria than previously published methods; as substrate serves a 1% tributyrin emulsion rendered permanent by the addition of one gram of Alkanol B per one hundred cc. That is a DuPont product which they kindly stated is alkyl-naphthalene sodium sulfonate. This emulsion is stable after putting it twice through a hand homogenizer and will keep at least one month if kept in the ice box. The only other reagents required are three dilutions of sodium bicarbonate: 1.00%, 1.11%, and 1.25%. The method is one of serial dilution and all analyses are done in duplicate. Ten tubes are set up in pairs to contain the following reagents: tube (1) receives 4 cc. of 1.25% NaHCO3; tube (2) 9 cc. of 1.11% NaHCO3; tubes (3), (4), and (5) 5 cc. of 1.00% NaHCO3. To tube (1) is added 1 cc. of the duodenal specimen to be analyzed and the same to tube (2). After thorough mixing 5 cc. of the contents of tube (2) are added to tube (3). In like manner 5 cc. of the contents of tube (3) are added to tube (4) etc. Five cc. are taken from the last tube of the series and discarded. In working with the duodenal contents a sixth tube is occasionally necessary and with experience one may predict that by noting the speed of digestion in the first tubes of the series immediately after the addition of the substrate. As soon as the various enzymic dilutions have been made, five cc. of the substrate are rapidly added to each tube. The tubes are then all well-stoppered, shaken and incubated in a water bath for two hours. The time interval begins the moment the substrate is added to the first tube and ends the moment the first tube is read in the colorimeter. The tubes should be read in the same order in which they are filled for within narrow limits it takes as long to fill them as it takes to read them in the colorimeter and in that way slight differences in the time of digestion for each tube are held to a minimum. Filter #565 is used for the colorimetric readings. Ninety-five per cent, of the light which it transmits lies in the range between 550 and 580 millimicra. For comparative purposes single figures expressing the relative enzymic effects of each specimen were obtained in the following manner: it was assumed that each tube exhibiting complete digestion, as shown by the reading in the colorimeter and the grossly apparent clarification of the tube's contents, had present 1% of undigested substrate. By using the equation for calculating the velocity constant of a first order reaction and expressing the time in minutes, a value for the constant of 0.03838 was obtained. This figure was multiplied by the reciprocal of the dilution of the specimen in the last tube of the series showing complete digestion and to that product was added the constant calculated from the Evelyn readings of the first tube in the series to show incomplete digestion. For clinical purposes it suffices to note the number of tubes in the series revealing complete digestion. The percentage of digestion of the substrate indicated by the colorimetric readings is determined by interpolation on a graph which represents the curve obtained by plotting the per cent, of undigested substrate against the colorimetric readings of standard dilutions of the substrate. For that purpose five tubes are prepared containing respectively 5, 4, 3, 2, and 1 cc. of the substrate diluted to a total volume, in each case, of 10 cc. with 1% NaHCO3. In this case, as in all determinations, 10 cc. of 1% NaHCO3 serves as a blank. The readings so obtained for different lots of emulsion do not vary by more than one quarter of a unit from the respective values: 5^0, 6^0, 7^2, 11^0 and 21^0 where the superscripts as usual indicate quarter units. The method just described was employed in the analysis of specimens obtained from fifty intubations. The subjects comprised ten hospital patients, twelve healthy nurses and twenty-one healthy medical students, of which latter group two were women. The activity in fasting specimens was found to range from zero to complete digestion through the fifth tube and partial digestion in the sixth. The specimens obtained at varying intervals, but usually fifteen minutes, after stimulation of the duodenal mucosa with twenty cc. of 0.1 N HCl displayed activity varying from complete digestion through the second tube with partial digestion in the third to complete digestion through the fifth with partial digestion in the sixth. The latter group showed a sufficiently narrow range of dispersion to permit statistical analysis on the basis of the number of tubes showing complete digestion. The 64 specimens analyzed after stimulation gave a mean of 3.34 tubes with a standard deviation of 0.82 and a probable error of the mean of 0.07. The chance of finding normal cases showing complete digestion only through the first tube was found to be 22 in 10,000. The analogous figure for complete digestion through the sixth tube was 6 in 10,000. The same figures for the group exclusive of the ten hospital patients (all of whom, however, gave results falling within normal limits but tending to fall in the lower range of normal) were: mean, 3.80; standard deviation, 0.72; and probable error of the mean, 0.07. On the basis of this limited group the chance of finding a normal case with complete digestion only through the first tube was found to be one in 10,000 and the chance of finding a normal case revealing complete digestion through the sixth tube was found to be eleven in 10,000. In fifteen cases at least two specimens obtained after stimulation were analyzed and in five of those, three specimens were analyzed. They usually showed complete digestion through the same number of tubes but in a few of the cases differed by one tube in that respect. It was concluded, therefore, that for clinical purposes the analysis of more than one specimen obtained after stimulation was not necessary. Seven subjects were twice intubated. Of those seven, three gave specimens after stimulation of the same strength on the two different occasions. Of the remaining four two gave results differing by only one tube; one differed by two tubes on the two occasions; the results in the seventh case cannot be compared as a neutral or faintly alkaline specimen after stimulation was not obtained after one of the two intubations. With the subject whose specimens differed by two tubes the first intubation, which gave the lowest value, took an exceptionally long time. The results indicate a tendency for specimens obtained from the same individual on different occasions to be closely parallel in enzymic activity. Reports in the literature indicate that such a test may have its greatest diagnostic value in cases of pancreatitis, steatorrhea, pancreatic and vicinal malignancies, pancreatic calculi and cysts and occasionally in biliary and hepatic diseases in which a secondary pancreatitis may be caused by the primary condition. The test may have prognostic value in following the course of a chronic pancreatitis.
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