FR2515828A1 - In vitro measurement of external pancreatic insufficiency - on duodenojejunal juice, by incubation with intrinsic factor and r-protein coupled to radioactivity vitamin=B12, then ppte. with antiserum - Google Patents

Abstract

In vitro measurement of external pancreatic insufficiency is effected on a sample of duodeno-jejunal juice, which has been clarified by centrifugation or other classical methods, and is then incubated in the presence of a mixture of free form intrinsic factor and R protein coupled with 57 Co-cyano-cobalamin and the total radioactivity (T) of the mixture is counted; when the incubation is finished an anti-R-protein serum and sodium sulphate mixture is added to the mixture and causes accelerated immunoprecipitation of the R-protein fraction coupled with non-degraded vitamin B12; the precipitate is centrifuged off and the radioactivity of the supernatant (S) is counted; the calculation P= Sx100/T allows determination of the percentage of vitamin B12 transferred by the R proteins to the intrinsic factor acceptor sites and provides a measure of the external pancreatic function. Determination of external pancreatic insufficiency, which takes account of physiological pancreatic digestion phenonomena and is very descriminating.

Description

Method for in vitro measurement, from a sample of duodenojejunal juice, of external pancreatic insufficiency, and reagents for the implementation of the method.

 There are many methods of functional exploration of the exocrine pancreas.

 The direct methods are based on assays of enzymes (amylase, lipase, proteolytic enzymes) in biological fluids: blood, stool, urine, intestinal fluid, pancreatic juice.

 Among the indirect methods, some call upon knowledge on the intraluminal transport of vitamin B12.

Recent work carried out by the team of
Professor ALLEN (DENVER-COLORADO) and also other studies carried out in vivo, have highlighted the important role of pancreatic proteases in the intraluminal transit of vitamin B12 and its acceptors.

 Indeed, it now appears clearly that vitamin B12 is preferentially combined with R proteins during the gastric stage of its digestive transit.

 In the distal duodenum and in the proximal jejunum, the R proteins are degraded by the pancreatic proteases, which releases vitamin B12 and allows its secondary transfer to the acceptor sites of the intrinsic factor: thus vitamin B12 can be normally absorbed at the ileal level . On the contrary, in external pancreatic insufficiencies, there is a defect in degradation of the R proteins, which prevents the transfer of vitamin B12 to the acceptor sites of the intrinsic factor. Vitamin B12 is therefore sequestered and the R proteins having no permeasive activity, vitamin B12 cannot be absorbed at the ileal level. This explains the existence of malabsorption in patients with external pancreatic insufficiency.

 To measure external pancreatic insufficiency, BRUGGE, ALLEN et al., Based on the lack of degradation of R proteins, have developed a modified Schilling test.

 It should be remembered that the Schilling test explores the intestinal absorption of vitamin B12 (not the external pancreatic function).

 Its principle is as follows: after ingestion of a low dose of radioactive vitamin B12, the body is saturated by intramuscular injection of a high dose of non-radioactive vitamin B12. Under these conditions, the absorbed radioactive vitamin B12 is eliminated in the urine.

 In the event of non-absorption, elimination is less than 5% of the ingested dose. In this case, the test can be completed by having the patient ingest labeled vitamin B12 and intrinsic factor (60 / ut). If the disorder is corrected, the lack of absorption is due to the deficiency of intrinsic factor characterizing Biermer anemia.

 If the elimination remains abnormally low, it can be concluded that there is a lack of absorption not linked to a deficit in intrinsic factor.

 The Schilling test should not be performed in the event of renal damage with glomerular filtration disorder.

 In the case of external pancreatic insufficiency, the R proteins which are not degraded sequester vitamin B12 which, not being transferred to the acceptor sites of the intrinsic factor during its intestinal transit, cannot be absorbed at the ileal level .

The BRUGGE, ALLEN et al. consists in making ingest
57 a combination of 57Co-cyanocobalamin coupled with the factor
58 intrinsic and Co-cyanocobalamin coupled with protein 58
A. Then we count the beta activity of Co and 1 gamma activity
57 of Co in 24-hour urine and expresses the results in relation to the two 58Co / 57Co activities. It is twisted from 0.03 to 0.09% in patients with pancreatic insufficiency and from 9.1 to 18.7% in normal controls.

 However, the preliminary study shows possible intermediate results in the two groups of subjects studied, in particular intermediate results in control subjects who present other affections concerning the intestinal transit (modification of the pH, imbalance of the intestinal flora, etc. ).

 This test therefore has the same drawbacks as the other indirect tests: it does not take into account the conditions of intestinal transit.

 In summary, each method of exploring exocrine pancreatic function, whether direct or indirect, is not sufficient, when used in isolation, to affirm the diagnosis of external pancreatic insufficiency. However, the reliability of direct methods is much greater than that of indirect methods.

 These methods, in addition to the production of casing, require, to be validly interpretable, the recourse to the measurement of several parameters and to stimulation. It would therefore be very interesting for the hospital clinical doctor to have a single direct parameter that does not require stimulation; it would be more easily interpreted and this would considerably simplify the production of pancreatic tubing, which would be better supported by the patient.

 The radioenzymological test according to the invention meets these objectives, because it accounts for a phenomenon of physiological pancreatic digestion and it is very discriminating.

The test according to the invention is an in vitro test carried out from a sample of duodeno-jejunal juice. The principle is as follows
After centrifugation and adjustment of the pH to 8, the supernatant of the duodeno-jejunal juice is incubated in the presence of a mixture of intrinsic pork factor in free form and of pork R protein coupled with 57Co-cyanocobalamin. The total radioactivity (T) of the bottle is counted (You can also use an intrinsic factor and R proteins other than pork).

During the incubation and the counting period, the pancreatic and intestinal enzymes degrade the R proteins; vitamin
B12 marked liberated binds to the acceptor sites of the intrinsic factor.

The addition of proc anti-protein R serum in the presence of sodium sulfate allows accelerated immunoprecipitation of the non-degraded protein R-vitamin B12 complex. After centrifugation and counting of the radioactivity of the supernatant (S) the percentage (P) of vitamin B12 transferred from the R proteins to the acceptor sites of the intrinsic factor is calculated: S x 100
T
The duodeno-jejunal juice is taken as follows
The patients studied are intubated using a 3-tube naso-gastrointestinal probe described by Professor NATUCHANSKI (CHU Poitiers), and modified in order to collect intestinal juice at the level of the second jejunal loop.

 The patients were fasted in the 24 hours preceding intubation. The progress of the probe is ensured by a ballast bag of mercury.

 The duodeno-junal fluid could also be obtained by aspiration during an endoscopy exploring the duodenum up to the angle of Thy.

An example of the procedure according to the invention is given below.
The different samples of intestinal juices collected are immediately placed on ice, then centrifuged; the pH is then brought to 8.

The reagents used for the test are the following a) an R1 reagent (one vial per test) containing intrinsic factor
freeze-dried pork, in free form (UB 12 BC = 7 ng of vitamin
B12 for example) and pig proteins R, freeze-dried
(UB 12 BC = 3 ng of vitamin B12 for example) coupled with
57Co-cyanocobalamin (for example 2 ng> radioactivity 10,000 cpm); b) an R2 reagent (one bottle per test) containing an anti-protein serum
R of pork, freeze-dried and capable of combining with a certain
amount of pork R proteins (having for example an activity
UB 12 BC 20 ng vitamin B12) and serum globulin
12
bovine (for example 5 mg) used as transporter c) a reagent R3 consisting of a 0.3 M phosphate buffer at pH = 7.4; d) a reagent R4 consisting of a 20% Na2SO4 solution.

The test procedure is as follows
Pipette duodenojejunal liquid (3 ml) previously centrifuged and adjusted to pH 8, directly into the bottle containing the reagent R1; incubate at room temperature for 1 hour, then count the total radioactivity (T) in the flask.

Then pipette successively into the above bottle
reagent R2 (1 ml)
reagent R4 (6 ml)
Stir, wait 15 minutes then centrifuge.

 The radioactivity (S) contained in the supernatant (2.5 ml) is then counted.

The percentage (P) of the labeled vitamin B12 transferred from the R proteins to the intrinsic factor is calculated.
S x 100 T
Results
In external pancreatic insufficiencies, there is a reduction in the degradation of the R proteins, or even an abolition of this degradation, and thus there is no transfer or an insignificant transfer of vitamin B12 from the R proteins to the acceptor sites. intrinsic factor.

 This is why the percentage P of this transfer is between O and 20% (O% P <20%).

 In normal subjects, the R proteins have been subjected to pancreatic enzymatic degradation and vitamin B12 is massively transferred to the acceptor site of the intrinsic factor, this is why the percentage of transfer is greater than 60 Z (60 ZCP <PC 100 %).

 The measurement method according to the invention thus allows a study of the overall proteolytic enzymatic activity, taking account of the enzyme-protein synergy, and measured on a physiological protein substrate. In addition, the parameter measured (percentage of degraded R proteins) is specific to the exocrine function of the pancreas.

Claims (5)

1. Method for in vitro measurement, performed on a sample of duodenojejunal juice, of external pancreatic insufficiency, characterized in that a sample of duodenojejunal juice previously clarified by centrifugation or other conventional means, is incubated in the presence of a mixture of intrinsic factor in free form and protein R coupled with 57Co-cyanocobalamin and that the total radioactivity (T) of the mixture is counted; once the incubation is complete, antiprotein R serum and sodium sulphate are added to the mixture, which causes accelerated immunoprecipitation of the fraction of protein R coupled with non-degraded vitamin B12, the precipitate is separated by centrifugation and counted. radioactivity (S) of the supernatant; the calculation of P = S xT100 makes it possible to determine the percentage of vitamin B12 transferred from the R proteins to the acceptor sites of the intrinsic factor, which constitutes a measure of the external pancreatic function. 2. Method according to claim 1, characterized in that the duodenojejunal juice samples were taken using a 3-tube naso-gastrointestinal probe described by MATUCHANSKI and modified so as to take intestinal juice at the second jejunal cove. 3. Set of reagents for implementing the method according to one of claims 1 and 2, characterized in that it comprises a) a reagent R1, in the form of doses for a test, consisting of  a mixture of intrinsic lyophilized pork factor and  57  lyophilized pork protein coupled to Co-cyanocobalamin b) an R2 reagent, in the form of doses for a test, consisting of  anti-protein R pig serum, lyophilized, capable of combining  with pig R protein, and bovine serum globulin c) a reagent R3 consisting of a 0.3 M phosphate buffer at pH = 7.4; d) a reagent R4 consisting of a 20% solution of Na2SO4. 4. Set of reagents according to claim 3, characterized in that each dose of reagent R1 contains a mixture of intrinsic pork factor having UB 12 BC activity: 7 ng of vitamin B12> and pig protein R having UB 12 BC activity: 3 ng of vitamin B12, coupled with 2 ng of 57Co-cyanocobalamin, the radioactivity of which is 10,000 c.p.m. 5. Set of reagents according to one of claims 3 and 4, characterized in that each dose of reagent R2 contains anti-protein R serum capable of combining with an amount of protein R having a UB 12 BC activity of 20 ng of vitamin B12 and 5 mg of bovine serum globulin.