CHEMICAL STUDIES ON INTESTINAL INTOXICATION.

I. THE PRESENCE AND SIGNIFICANCE OF HISTAMINE IN AN OBSTRUCTED BOWEL.

BY R. W. GERARD.

(From the Hull Physiological Laboratory of the University of Chicago, Chicago.)

(Received for publication, December 13,192l.)

That the systemic symptoms and death following an acute intestinal obstruction are due to the absorption of toxins from the obstructed part of the bowel has been established by the consistent results of all recent workers in this field.

The closed loop operation introduced by Whipple and his associates (1, 2) has shown that these toxins may be developed in- dependently of foods or the special secretion of any one part of the enteric canal; so that they must arise from the mucosa and its secretions or the bacteria in the lumen of the gut.

The bacteria of the normal gut multiply greatly in the closed gut (3) and the proteolytic strains completely overgrow all others (4) ; decarboxylating organisms, mainly of the coli group, which produce histamine under conditions that prevail in a closed loop, have been isolated from feces and intestinal contents (2, 5, 6, 7) ; and histamine has been found in normal feces (4, 8) and mucosa (9). True exotoxins are not present in loop fluid (10, ll), so that the amine (or proteose) nature of the toxic agents is strongly indicated.

The loop toxin is absolutely identical with histamine in its pharmacologic action (12, 13, 14), except that it renders blood incoagulable; and, also like it, resists the action of heat (15, 16, 17, IS), acid (refluxing with 20 per cent HCl)’ alkali, trypsin, and erepsin (2, 19, 20, 21). When loop fluid is treated with alcohol or

1 Personal communication from Dr. L. R. Dragstedt confirmed by the author. He found the minimum lethal dose for guinea pigs to increase from 3 to 5 cc. after the acid treatment.

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112 Studies on Intestinal Intoxication. I

half saturated with ammonium sulfate, part of the toxicity appears in the precipitate, as would a proteose, and part remains in the filtrate, as would an amine.

Nesbitt (22) found choline and neurine in obstruction fluid after feeding yolk. Kukula (15) isolated a pentamethylene diamine (cadaverine?) from fluid above an obstruction. Roger (23) showed a proteose present in similar material, and Whipple later secured evidence of a primary heteroproteose (24). Sweet, Peet, and Hendrix (25) found in obst#ruction fluid a proteose and a “choline-like” substance. Taylor and Pearce (26) were unable to demonstrate histamine in loop fluid by Kutscher’s tannic acid method, possibly because the amine was carried down in the tannic acid precipitate-as in solutions of low acidity.2

In view of its importance, analysis of obstruction fluid for his- tamine by more adequate methods was undertaken in this in- vestigation.

Methods.

Histamine was tested for in loop fluids by chemical analysis, by biological assay on strips of intestine, and by intravenous in- jection in dogs. For chemical analysis the quantitative method of Koessler and Hanke (27, 28, 29, 30) and Koch was used. This consists, briefly, of acid hydrolysis of the protein ‘material, dis- tillation of ammonia, precipitation of the amines and amino- acids with acid phosphotungstic acid, decomposition of this precipitate with baryta, concentration, addition of NaOH to the concentrate, and repeated extraction of the alkaline solution with amyl alcohol to separate the histamine from histidine. Any interfering substances still present are removed by treatment with AgNOs and baryta. The histamine is tested for calorimetrically by coupling with diazobenzene sulfonic acid. As little aa 0.001 mg. of histamine dihydrochloride (in 8 cc. of test mixture) may be determined quantitatively in this manner.

Guggenheim and LGffler (31) showed the intestine of the guinea pig affords a very satisfactory test for ‘histamine. Longitudinal pieces suspended in warm oxygenated Ringer’s solution respond to

SMeakins and Harington (Meakins, J., and Harington, CL R., J. Pharmacol. and Exp. Therap., 1922, xviii, 455) have just reported finding histamine in human obstructed colons.

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R. IV. Gerard 113

concentrations of histamine as low as 1 part to 250 million. In using this test, the test solutions were carefully neutralized and any preservatives removed, as these factors modify the contrac- tion of the gut.

Injections in the dog were made directly into the femoral vein. Blood pressure was taken from the carotid artery.

EXPERIMENTAL.

Histamine Present in Fluid Accumulated in Closed Washed Loops of Jejunum.

Sample 1.-A closed washed loop of the jejunum was made and con- tinuity of the gut reestablished by end to end anastomosis. The dog ran the usual course and died in a typical manner. 90 cc. of chocolate brown fluid with a putrid odor were collected at autopsy and at once boiled. 6 gm. of dark brown solid were obtained by evaporation. 1 gm. of the powdered solid was refluxed (just kept simmering) for 100 hours with 20 per cent HCl. Considerable carbonization occurred despite careful heating, and a large amount of gummy melanoid material which was refrac- tory to the further treatment was formed. The hydrolysate was distilled twice in uacuo, filtered from the melanoid matter, slightly acidified with HCl, evaporated, and precipitated with phosphotungstic acid, etc., as indicated above. By double extraction with amyl alcohol both a histidine fraction and a histamine fraction (Solution 1C) were obtained, which were diluted, transfered to precision cylinders, and tested. 1 cc. of Solution 1C gave a color value of 1.1 mm., corrected, which equals 0.9014 mg. of histamine dihydrochloride (by table). The number of mg. of histamine

dihydrochloride per 100 cc.. of original &id equals 0.0014 X 25 X 151.2 X

6 X g equals3 0.6 mg.

Solution 1C was further tested for histamine by its action on guinea pig gut. 4 cc. were added to the 50 cc. of oxygenated Ringer’s solution in which the intestine was suspended. The concentration of histamine in this solution, as given by the calorimetric test, is 1:10,000,000, and the contraction of the gut following its addition was similar to that following the addition of known histamine in like quantity.

Solution 1 C also caused a fall in the blood pressure of a dog when injected intravenously. The shape of the curve was identical with one produced by the injection of known histamine solutions.

*Total volume of Solution 1 C equals 25 cc. 15.2 7 X PI represents

dilutions in process of analysis, 1 gm. of solid was equivalent to 15 cc. of the original fluid.

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114 Studies on Intestinal Intoxication. I

Histamine Removed by Melanin.

Sample K-10 mg. of histamine dihydrochloride were added to 1 gm. of Solid 1, and the whole analyzed parallel with it as a check upon it. Carbon- ization and melanin formation occurred during acid hydrolysis just as with Sample 1. Only about half (4.4 mg.) of the histamine dihydrochloride added was recovered in the final fraction.

The method used had proved quantitative when tested on pure imidazole solutions, histamine is not easily destroyed even by vigorous chemical treatment (28), and Hanke and Koessler (30) report that histamine is adsorbed by charcoal. In this sample t.he charred melanoid material formed during hydrolysis removed the histamine by adsorption. (Further evidence for this is afforded by Sample 5.) In Sample 1 also, a considerable quantity of histamine was probably removed in a like manner.

Alkaline Hydrolysis of Loop Fluid Does not Permit a Quantitative Determination oj Histamine.

Sample 6.-10 cc. of fresh jejunal loop fluid were allowed to stand with 3 gm. of NaOH, and the mixture was extracted with amyl alcohol and sul- furic acid, neutralized, dried, and tested, as above. A similar portion with 1 mg. of histamine dihydrochloride added was treated in like manner. In both cases a large amount of brown material was formed and the amyl alcohol extracts were deeply colored (reddish brown). The color did not pass into the acid. Both samples gave tests for histamine but not quantitatively.

Presence of Histamine in Loop Fluid Confirmed by Further PurijZcation.

Sample ,??.-A closed washed loop of the ileum was made and continuity of the intestine reestablished as before. At autopsy 130 cc. of reddish brown fluid were collected and at once evaporated to dryness. 7 gm. of dark brown solid were formed. 2 gm. of this were subjected to acid hydrol- ysis, etc., exactly as was Sample al. The histamine fraction was tested calorimetrically.

0.5 cc. gave a color test of 4 mm., corrected, which equals 0.55 mg. histamine dihydrochloride for the sample taken.

1.0 cc. gave a color test of 6 mm., corrected, which equals 0.40 mg. hista- mine dihydrochloride for the sample taken.

The average of these figures taken showed 1.3 mg. of histamine dihydro- chloride per 100 cc. of original fluid.

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R. W. Gerard 115

As pointed out by Koessler and Hanke (27), the failure of twice the quantity to give twice the color value indicated interference with the coupling reaction by some substance. A further purification with AgNOa and baryta was resorted to, and histamine again demonstrated in the silver-insoluble portion by all tests, as in Sample 1.

In the above analysis all the discarded fractions were tested for toxic material by intravenous injection into a dog. None of these fractions caused a fall in blood pressure.

Histamine in Loop Fluid Determined Quantitatively.

The above analyses show only the minimum quantity of his- tamine, for much is removed by the melanin formed in hydroly- sis. To avoid formation of this the undried fluid was used for hydrolysis. The following group of analyses done in this w’ay yielded quantitative results.

Sample 7.-200 cc. of chocolate brown fluid were obtained at autopsy from a closed jejunal loop of 3 days standing. This was centrifugsd to remove solid particles and the opalescent supernatant liquid used fol. this test, 10 cc. were at once refluxed with 37 per cent HCI for 40 hours (Sample 7A). No gummy material formed. A second 10 cc. portion was similarly treated after the addition of 10 mg. of histamine dihydrochloride (Sample 7B); and the hydrolysis again accomplished without carbonization. The subsequent treatment paralleled that of Sample 1. 1 cc. of the final hista- mine fraction for Sample 7A gave a color test of 15.7 mm., corrected, which by table corresponds to 0.021 mg. of histamine dihydrochloride, or 0.3 mg. of histamine dihydrochloride for the original 10 cc. sample. The final fraction of Sample 7B was so concentrated that 0.05 cc. gave a color test of 23.5 mm., corrected, which equals 0.0313 mg. histamine dihydrochloride, or 9.8 mg. for the whole sample taken. 0.3 mg. originally present in the fluid is included here, so 9.5 mg. of the added histamine dihydrochloride were recovered. This is 95 per cent of the total; and is within experimental error for a concentrated solution.

The solid thrown down by centrifugalization was roughly analyzed for histamine to eliminate any loss by occlusion. The solid was suspended in 25 cc. of water and 10 cc. used for extraction with amyl alcohol (3 portions of 20 cc. each) and this in turn with N HISO, (3 portions of 10 cc. each). The combined acid extracts were treated as usual. 1 cc. of the histamine fraction had a color value of 4.1 mm., corrected, which equals 0.0055 mg. of histamine dihydrochloride, or 0.2 mg. for the whole sample of loop fluid. There was present in the200 cc. of fluid, therefore, at least the equiv- alent of 6.2 mg. of histamine dihydrochloride.

These various histamine fractions gave the typical depressor effect when injected into dogs.

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116 Studies on Intestinal Intoxication. I

Sample O.-A closed loop of the upper jeju,num was made by the usual operation. 44 hours later the dog was very ill and could not stand. It was sacrificed, and 40 cc. of bloody brown foul smelling fluid were obtained from the distended loop. 15 cc. of this were at once refluxed for 40 hours with 37 per cent HCl, and the further procedure of Sample 1 was followed. The histamine fraction finally secured was tested in the usual.way. 1 cc. gave a color value of 18.5 mm., corrected, which equals 0.025 mg. of hista- mine dihydrochloride, or 0.3 mg. for the sample taken. This would corre- spond to 0.8 mg. for the original fluid obtained. This fraction also gave a typical histamine depression of blood pressure.

Sample 10.-A closed loop of the upper jejunum was made as usual. The dog became progressively weaker and died in 4 days. 125 cc. of bloody brown foul smelling fluid were obtained at autopsy. 15 cc. were at once refluxed with acid and carried through the usual procedure. The hista- mine fraction finally obtained was tested calorimetrically. 1 cc. gave a color value of 23 mm., corrected, which equals 0.031 mg. of histamine dihydrochloride, or 0.4 mg. for the sample taken. The total fluid obtained contained 3.3 mg. of histamine dihydrochloride.

In the above analyses the time of appearance and shade of the color were correct for histamine.

Histamine Present in Material Accumulated in Closed Washed Loop of Colon.

Acute obstruction in the lower bowel, especially the colon, is not attended by such a fulminating syndrome as is higher ob- struction. Many human cases of obstipation lasting for months are known; and closed loops of the colon in dogs have been found by many workers (32, 33, 34) to be entirely compatible with life. In such loops of colon, however, a fecal-like mass accumulates which is extremely toxic and, upon intravenous injection in dogs, shows a typical depressant action. The following analysis shows the presence of histamine in such material.

Sample 8.-A closed loop of the colon was made in the usual manner.’ The dog made an uneventful recovery and showed no symptoms for 8 months, when it was again operated upon and the closed loop removed. It contained 100 gm. of a grey buttery solid. This was triturated with water and the suspension found to give a typical loop toxin fall in blood pressure upon injection. 10 cc. of the suspension (equals 1 gm. of solid) were refluxed with 37 per cent HCI for 45 hours. Subsequent treatment as above yielded a histamine fraction, 1 cc. of which gave a color value of 7.5 mm., corrected, which equals 0.01 mg. of histamine dihydrochloride, or 0.14 mg. for the sample taken. There was present in the original sample, therefore, at least 14 mg. of histamine dihydrochloride.

4 Operation performed by Dr. L. R. Dragstedt.

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R. W. Gerard 117

Histamine Present in Sterile Jejunal Mucosa but Not in Sterile Jejunal Secretion.

The aseptic formation of hi&amine in the body has not been conclusively demonstrated. Barger and Dale (9) found this amine in washed ox mucosa which had, however, been harboring proteolytic bacteria. The evidence presented by Abel and Kubota (35) favoring the formation of histamine during normal metabolic processes, has been questioned by Hanke and Koessler (36).

Much evidence indicates that the obstruction tosins are in part, at least, the result of bacterial activity and formed only in their presence (18, 33, 37, 38, etc.). The following experiment affords further evidence on these points.

Sample 11 .-The usual operation for a closed loop of the jejunum was performed, and an end to end anastomosis made. The isolated segment of gut attached only by its mesentery, was not closed by infolding the ends however, but left open. Further exposure of the mucous surface (to the sterilizing action of the peritoneal secretions, 33) was secured by a slit along the antimesenteric border of the loop, which allowed it to evert. The mucosa was well washed with sterile water, rinsed with ether, and dropped back into the abdomen, which was then closed. The dog made a perfect recovery and remained normal. 149 days later she was still entirely normal (3 days before being sacrificed she was observed in coitus), and was sacri- ficed. The autopsy was performed immediately under aseptic conditions. The peritoneum and mesenteries were moderately congested and were besprinkled with fibrin-like granulations, though no adhesions between them were found. The open loop was adherent about its edges but the mucosa was freely exposed in the cavity. The mucosa appeared entirely normal on gross and microscopic examination. Within the peritoneal cavity were about 200 cc. of light grey mucilaginous material, the accumu- lated secretion of the everted mucosa. Cultures of the mucosa and secre- tion taken on agar, blood agar, and Endo plates, agar slants, and in broth were all negative. The whole abdomen presented a strikingly pathological picture, yet the dog was in good health when sacrificed.

10 gm. of the mucous secretion were at once refluxed with 40 cc. of 20 per cent HCl for 33 hours, and the usual analysis was carried out. 1 cc. of the final histamine fraction gave a color value of about 15 mm. but the color was distinctly green. Intravenous injection into a dog of the original mate- rial, or of the histamine fraction, had no effect on blood pressure. Hista- mine was therefore absent from the sterile jejunal secretion.

The mucosa of this sterile loop was thoroughly washed, scraped off, minced, and at once refluxed with 25 cc. of 20 per cent HCl for 33 hours. Its moist weight was 6 gm. The usual analysis was performed and the final histamine fraction secured. 0.25 cc. of this solution gave a color

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118 Studies on Intestinal Intoxication. I

value of 14 mm., corrected, which equals 0.019 mg. of histamine dihydro- chloride, or 10 mg. for the sample taken. On intravenous injection into a dog this solution gave a typical histamine depression similar to one pro- duced by known histamine of like strength.

The sterile mucosa of the dog’s jejunum, therefore, may contain histamine. No explanation is offered. The experiment is being repeated to determine t,he constancy of this condition.

Histamine Present in the Mucosa of a Closed Jejunal Loop.

The r81e of the intestinal mucosa in the formation of loop toxin haa been especiallly emphasized by Whipple, who believes the toxin to be a perverted secretion (21). The presence of his- tamine in loop mucosa as shown below is of interest in this con- nection, and aa a check on the above.

Sample IO.-The mucosa of Dog 10 in the above series was used. At autopsy the closed loop was found distended but not perforated. The mucosa was somewhat injected, but otherwise normal. The ends which had been infolded and tied were gangrenous. The end to end anastomosis showed a small perforation, and a hemorrhagic peritonitis had developed.

The mucosa was carefully washed free of all contents and partly dried. 6 gm. were refluxed with 37 per cent HCl, and carried through the remaining procedure as above. The final histamine fraction was tested as usual. 1 cc, gave a color value of 15 mm., corrected, which equals 0.02 mg. of hista- mine dihydrochloride, or 0.3 mg. for the whole sample.

Hi&dine Present in Loop Fluid and Mucosa.

The histidine content of some of these samples was determined in the manner indicated under the analysis of Sample 1. Follow- ing are the results.

Sample 2 contained 78 mg. of histidine dihydrochloride per 100 cc. of fluid, or 101 mg. for the entire quantity.

Sample 8 contained 1.5 mg. of histidine dihydrochloride per gm. of solid, or 150 mg. for the entire quantity.

Sample 9 costained 73 mg. of histidine dihydrochloride in 100 cc. of fluid, or 29 mg. for the entire quantity.

Sample 11 (mucosa) contained 2.4 mg. of histidine dihydro- chloride for the entire 6 gm. of moist mucosa.

Sample 11 (fluid) contained 14.1 mg. of histidine dihydrochlor- ide in 10 gm. of jelly, or 280 mg. for the entire quantity.

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R. W. Gerard 119

Sample 10 (mucosa) contained 31.4 mg. of histidine dihydro- chloride for the entire 6 gm. of moist mucosa.

Sample 10 (fluid) contained 30 mg. of histidine dihydrochloride in 100 cc. of fluid, or 37 mg. for the entire quantity.

Such widely varying figures obtained under dissimilar condi- tions are too few to permit any generalizations, though it might be noted that Sample 11 (mucosa) which was rich in histamine is poor in histidine. In all cases: however, there is present an ample supply of histidine to account for the presence of the his- tamine if the latter were entirely derivea from it by decarboxyla- tion. The sequence is thus practically complete; from the pres- ence in closed loops of histidine-rich substrates and histamine- forming bacteria, to the actual isolat,ion of the histamine so formed. Since histamine is present in the normal gut, obstruction must involve an increased production of this amine, a change in its absorption or detoxication by the mucosa, or all these factors; else no systemic symptoms could appear.

Histamine is probably only one of the amines formed by protein decomposition and is more important only in that it is the most active. The other putrefaction products must all contribute their toxicities to the total.

Combined Histamins also Present in Loop Fluid. Evidence for a “Peptamine Histamine.”

Proteosea as well as amines may play a significant part in the obstruction toxemia. A toxic proteose fraction has been isolated from loop fluid by Whipple and his collaborators (24), which re- sembles loop toxin in activity, including the anticoa.gulant action. Also it is alcohol-insoluble, as part of the loop toxin is. Certain properties, however, the two do not appear to possess in common, for no proteose can resist the chemical treatment which the loop toxin withstands, for example boiling with 20 per cent HCI. These facts would all be brought into harmony by the presence of a histamine proteose of the nature of the peptamines synthesized by Guggenheim (39), and which gave a histamine-like action.

Bacteria having the power of decarboxylating free histidine might well attack the exposed carboxyl group on a histidine molecule attached by its imidazole group to a protein chain. A compound, such as would be formed by this process, should be

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120 Studies on Intestinal Intoxication. I

alcohol-insoluble, non-diffusible through collodion, yield histamine upon hydrolysis, and show a histamine-like pharmacodynamic action with a peptone-like action (e.g. anticoagulant) super- imposed. Acid hydrolysis would not materially decrease its toxicity, as the histamine so liberated would compensate for the proteose -linkings destroyed. These are exactly the properties of one of the loop toxins.

The red dye used as a test for histamine is formed by the coup- ling of the diazotate (or its tautomeric nitrosamine) with the imidazole nucleus. The ‘peptamine above postulated would have this nucleus combined with the peptide chain, and should give a feebler color test for histamine than would the hydrolysate. This difference between free and combined histamine, and their different alcohol solubilities, make possible the following analy- sis for the peptamine form.

Sample 5.-5 mg. of histamine dihydrochloride were added to 50 cc. of fresh fluid from a closed loop of jejunum, and the whole was boiled. Coag- ula which formed were removed by centrifuging the whole after 24 hours; washed; and the washings added to the original red opalescent supernatant liquid. Over half the added histamine dihydrochloride was carried down in the coagulum, as only 2.4 mg. were subsequently recovered. This shows that the losses in the early experiments, recorded above, were indeed due to adsorption. The fluid and washings (45 cc.) were treated with5 volumes of 95 per cent alcohol and the whole was allowed to stand over night. -4 gummy orange precipitate formed, which was filtered from the alcohol solution and washed. The alcohol filtrate was clear and of a mahogany color. Analysis showed this to contain all the free histamine recovered.

The alcohol precipitate was extracted with boiling water, and the solu- tion divided into 2 parts. One was at once extracted with amyl alcohol and acid, and the final histamine fraction tested calorimetrically. There uus no color formed. The other part was hydrolyzed by refluxing for 70 hours with 20 per cent HCl and then distilled in V~CUO twice as with Sample 1. The residue from the second distillation was extracted with amyl alcohol and acid. The histamine fraction finally obtained gave a color test corre- sponding to 0.6 mg. of histamine dihydrochloride for the alcohol precipitate. This precipitate, therefore, contained no free histamine before hydrolysis but did contain some after hydrolysis.

Sample 7.-A portion of this was tested by Koessler and Hanke’s method for histamine, as detailed above. 30 cc. of the remainder were treated with 5 volumes of 95 per cent alcohol and allowed to stand over night. A precip- itate was removed by filtration, and suspended in water (Fraction 7PS). This caused a slight drop in blood pressure on intravenous injection, but not so marked as that caused by the alcohol filtrate (Fraction 7FA) which contained the free histamine (See Fig. 1).

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R. W. Gerard 121

Part of the suspension (Fraction7PS) was at once extracted and a histamine fraction secured. This gave a slightly green color test which, if all due to histamine, would correspond to 0.25 mg. for the whole alcohol precipitate. The remainder of the suspension was hydrolyzed by refluxing with20 per cent HCl for35 hours,.and then treated as the similar fraction in Sample 5. The final histamine fraction gave a color value corresponding to 1.0 mg. of histamine dihydrochloride for the whole alcohol precipitate, or 0.75 mg. more than before hydrolysis. This sample, therefore, also shows the presence of combined histamine.

FIG. 1. Typical tracing. Dog. Ether anesthesia. Carotid blood pres- sure. 1 1 = injection of 1 cc. of the alcohol filtrate of Fluid 7 (Fraction 7FA). 5 2 = injection of 1 cc. of a water solution of the alcohol precipitate of Fluid 7 (Fraction 7PS). 2 cm. = 1 min.

SUMMARY OF RESULTS.

Table I shows the presence of histamine in seven out of eight loop fluids analyzed. The fluid that contained none had been formed under sterile conditions and had caused no symptoms. The presence of histamine in sterile as well as loop mucosa, however, forbids the conclusion that this amine can be formed on& through the agency of bacteria. The presence of a peptamine histamine is shown for two samples of loop fluid. The chemical properties and biological action of closed loop fluids may be accounted for by their content of free and combined histamine, though other toxins surely are present.

CONCLUSIONS.

1. Histamine is present in the contents of isolated closed loops of the large or small intestine. The amount varies in different samples; 2 to 3 mg. of histamine dihydrochloride per 100 cc. of fluid being a fair minimum average.

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122 Studies on Intestinal Intoxication. I

Sample of Total loop fluid. volume.

9 10 11 10 Mucosa.

11 “

cc.

90 130

200

100 km.)

40 125 200

6 km.)

6 km.1

TABLE I.

r0td ~0nt.3 of histidme dih$rghlw

m0.

101

15

29 37

280 31

2.4

c-& oy hi&id& dihy

drochloride.

w.

98

15 (per 10 gm.

73 30

140 52

(per 10 gm. 4

(per 10 gm.

‘otal oonten Df histamine dih$t&phlo-

w.

0.5 1.7

Plus. 6.2

14 .o

0.8 3.3 0.0 0.3

10.0

“;;pEtff= &tam& dihy-

drochloride.

m&l.

0.6 1.3

Plus. 3.1 1.4

(per 10 pm.) 2.0 2.7 0.0 0.5

(per 10 pm.) 16.7

(per 10 pm.)

Sample of loop fluid. Total combined histamine in alcohol-insoluble portion. Histamine dihydrochlorida.

5 7

mu. 0.6 (from 50 cc. of fluid.) 0.75 (from 30 cc. of fluid.)

2. A combined histamine derivative is also present in obstruc- tion fluid. Evidence is presented that this is of peptide nature.

3. The sterile secretion of jejunum contains no histamine (1 experiment.).

4. Sterilemucosaof the jejunum contains histamine (1 experiment) ; as does the mucosa of closed loops of the jejunum (1 experiment).

5. Histidine is present in loop fluid and mucosa. 6. Histamine is carried down in heat and acid coagula of loop

fluid, and small amounts may be adsorbed in the alcohol precipi- tate from loop fluid.

The writer wishes to express his indebtedness to Professors A. J. Carlson and L. R. Dragstedt for much valuable aid and caunsel throughout this work, to Professor F. C. Koch for pure histidine dihydrochloride kindly supplied by him and for other kindnesses, and to Drs. M. T. Hanke and K. K. Koessler for

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R. W. Gerard 123

the gift of 1 gm. of histamine dihydrochloride and other assist- ance. The experimental work has been much facilitated by the able assistance of Mr. Harold Klawans.

BIBLIOGRAPHY.

1. Stone, H. B., Bernheim, B. M., and Whipple, G. H., Bull. Johns Hopkins Hosp., 1912, xxiii, 159.

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3. McClure, R. D., J. Am. Med. Assn., 1907, dlix, 1003. 4. Cannon, P. R., Dragstedt, L. R., and Dragstedt, C. A., J. Infect. Dis.,

1920, xxvii, 139. 5. Meilanby, E., and Twort, F. W., J. Physiol., 1912-13, xiv, 53. 6. Berthelot, A., and Bertrand, D. M., Compt. rend. Acad., 1912, cliv, 1643,

1826; 1912, clv, 360; 1913, clvi, 1027. Berthelot, A., Compt.rend. Acad., 1913, clvi, 1567. Bertrand, D. M., and Berthelot, A., Lancet, 1913, i, 523.

7. Jones, H. M., J. Infect. Dis., 1918, xxii, 125. Hanke, M. T., and Koessler, K. K., J. Biol. Chem., 1922, 1, 131.

8. Eustis, A., Am. J. Med. SC., 1912, cxliii, 862. 9. Barger, G., and Dale, H. H., J. Physiol., 1910-11, xli, 499.

10. Dragstedt, 0. A., Dragstedt, L. R., and Chase, C. S., Am. J. Physiol., 1918, xlvi, 366.

11. Dragstedt, C. A., and Moorhead, J. J., J. Exp. Med., 1918, xxvii, 359. 12. Dale, H. H., and Laidlaw, P. P., J. Physiol., 1910-11, xii, 318; 1911-12,

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R. W. GerardAN OBSTRUCTED BOWEL

AND SIGNIFICANCE OF HISTAMINE ININTOXICATION: I. THE PRESENCE

CHEMICAL STUDIES ON INTESTINAL

1922, 52:111-124.J. Biol. Chem. 

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