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gastric fistula. The secretion of hydrochloric acid and pepsin was measured before and after the rats had been burned. After burns, a smallbut not sig-.
Br. J. exp. Path. (1973) 54, 665

HISTAMINE METABOLISM AND GASTRIC SECRETION IN EXPERIMENTAL BURNS IN THE RAT S. E. SVENSSON AND H. WETTERQVIST From the Departments of Physiology and Clinical Physiology, University of Lund, Lund, Sweden Received for publication July 30, 1973

Summary.-The urinary excretion of histamine and the histamine formation in vitro in burned and control male and female rats were measured. With increasing temperature, the histamine excretion increased in male and female rats. The histamine formation in vitro was increased in burned skin. The changes are presumably a result of the inflicted injury, which means that in the rat histamine is involved in the inflammatory reaction. A small and probably significant reduction in histamine forming capacity was observed in the stomach from burned and starved female rats. Female rats were provided with a gastric fistula. The secretion of hydrochloric acid and pepsin was measured before and after the rats had been burned. After burns, a small but not significant reduction in gastric secretion was seen. It is proposed that the burn injury in some way or another may inhibit the gastric function for a while.

THE URINARY excretion of histamine is increased after burns in mice (Dekanski, 1945), dogs (Kisima, 1938), rats (Sanyal, 1962; Wetterqvist and White, 1968) and man (Birke et al., 1957; Tham, 1966; Agrup et al., 1973). The mechanism behind this increase is still obscure. By measuring the urinary excretion of total histamine and methylhistamine as well as radioactive histamine and methylhistamine after repeated subcutaneous injections of radioactive histidine, Wetterqvist and White (1968) found increased histamine formation in burned rats. This paper contains further observations on the influence of temperature on histamine exeretioni. The site of increased formation of histamine in mice has been shown to be the skin (Schayer and Ganley, 1959), but even the lung and liver are involved (Johansson and Wetterqvist, 1963). In this study the rate of histamine formation was determined in the burned skin and gastric mucosa. The latter tissue was considered essential since it seems to be the main contributor to urinary histamine (High, Shepherd and Woodcock, 1965) and it has been shown that increased formation of histamine is linked to the excitation of hydrochloric acid secretion (for references, see Kahlson, Rosengren and Svensson, 1972). Observations on this aspect might throw light on the finding of duodenal ulceration in extensive burns in humans (Curling, 1942; Harkins, 1938). Therefore, gastric secretion in connection with induced burns was also studied. MATERIALS AND METHODS

Animals.-IMale and female rats of the Sprague-Dawley strain (weight 200-275 g) were

used. They were fed a constant amount of a semisynthetic diet essentially free from histamine (less than 0-02 ,g/g) (Kahlson, Rosengren and Westling, 1958). Tap water was freely available.

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S. E. SVENSSON AND H. WETTERQVIST

Induction of thermal injury.-The rats were anaesthetized with ether. A standardized area of the back skin was exposed to water at 400, 500, 600, 70° or 800 for 25 seconds. Immersion in water at 600 or above induced a burn injury with an elliptic area of about 6 x 8 cm. In rats subjected to gastric secretion studies water at 370 was used in control experiments. Determination of urinary histamine.-The rats were kept in metabolism cages and the urine was collected under hydrochloric acid in 24-hour portions. The excretion of free histamine was determined by bioassay of the diluted urine on the guinea-pig ileum, as described by Angerwall, Bjuro and Westling (1961). Values are given as,ug of free histamine base excreted per 24 hours. The identity of the compound investigated was checked by antihistamine as described by Reuse (1948). The animals in cages were injected subcutaneously once daily with aminoguanidine in a dose of about 20 mg/kg of body weight in order to inhibit the activity of histaminase, thus increasing the urinary excretion of free histamine. Determination of histamine formation in vitro.-Determining urinary histamine will obviously not indicate the site at which changes in the rate of formation take place. This information can be obtained by excising tissues and examining in vitro their histidine decarboxylase activity. Female rats were starved for 12 hours before being burned. After inflicting the burn injury, one half of the animals were given food once during 24 hours or twice during 48 hours. The other half received no food or water. This means that animals killed 48 hours after being dipped into water had been starved during 60 hours. Histidine decarboxylase activity of the skin of the back and the glandular portion of the stomach was determined in vitro with a modification of Schayer's technique (Kahlson et al., 1958). The amount of [14C] histamine formed from [14C] histidine by minced tissue was determined after purification and crystallization as pipsylhistamine. The method has been described in detail by Bjur6, Westling and Wetterqvist (1961). Perchloric acid was used instead of trichioroacetic acid in order to precipitate the proteins. Histidine decarboxylase activity is given as ct/min of [14C] histamine formed by 1 g of tissue in 3 hours in vitro, with blank values and background radioactivity subtracted. [14C] radioactivity was measured at infinite thickness under standardized conditions in a flow counter with a background of 31-35 ct/min. [14C] 1-histidine was obtained from the Radiochemical Centre, Amersham, England, labelled in the 2 position of the imidazole ring, 1 ,ug of [14C] histamine formed from this ["4C] 1-histidine would give about 6800 ct/min with 40 mg histamine base as carrier and assayed as above. [14C] 1-histidine usually contains a small amount of radioactive histamine as an impurity. This was removed almost completely by use of a Dowex 50 column, as described by Kahlson et al. (1963). Collection of gastric juice.-Female rats were provided with a gastric fistula according to Lane, Joy and Joy (1957). About 4 weeks were allowed for recovery from surgery before experiments were begun. Gastric juice was collected by simple drainage from the gastric fistula. The gastric juice was analysed on hydrochloric acid secretion by titration with 0-1 N NaOH with phenol red as indicator. Peptic activity was estimated mainly according to the method of Hunt (1948). The values are expressed in ug pepsin by comparing the proteolytic activity in gastric juice with a preparation of crystallized hog pepsin from Sigma Chemical Company, St Louis 18, Missouri, U.S.A. (Batch 95 B-1270). The method has been proposed by Bitsch (1966). Test procedure.-Interdigestive secretion was collected for 11 hours after the stomach had been gently washed 3 times with body-warm 0O9% NaCl solution for one hour. The interdigestive period was followed by a subcutaneous injection of 0-4 ug gastrin II and collection of the gastric juice for one hour. This procedure was performed on 4 different days: (a) 4 days before b; (b) 18-22 hours after exposure of the rats to water at 370; (c) 18-22 hours after the induction of experimental burns with water at 600; 4 days after (b); (d) 5 days after burning. RESULTS

Histamine excretion In the male rats (Fig. 1) there is a decrease in urinary histamine on the day of being dipped into water at 600 or more. Using water at 700 or 80° the excretion

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HISTAMINE METABOLISM AND GASTRIC SECRETION IN THE RAT

669

of histamine increases during 48-72 hours. During the following days the urinary histamine is still excreted in amounts exceeding those before the burn. In female rats (Fig. 2) water at 600 induces a slight but obvious increase in the histamine output during the following 48-72 hours. Water at 700 or 800 further increases the histamine excretion, which after that does not return to the values before the burn. Histamine formation The formation of histamine in vitro is significantly increased in the dorsal skin covered by the burn (Table I) compared with the controls (P < 0'01 for 3 of the 4 groups and P < 0-05 for the last group; starved 48 hours after the burn).

TABLE I 370

600

Stomach

Skin

Stomach

Skin

16930±3840

5±15

Food

14650±4420 180±90

No food Food

3360±440 240+100 7660±3360 2±5 18690±3440 340±110 21820±2080 20±25

24 hours

48 hours

No food 1580±680 470±-320 1840±650 50±40 Histamine formation in vitro in back skin and stomach from burned and control rats, fed or starved. The values are mean values from 4 or 5 rats + standard deviation and are expressed in ct/min of [14C] histamine formed from [14C] 1-histidine per g tissue. Background and blank values are subtracted.

In the stomach there is a tendency towards lower values in burned rats, fed or starved, compared with control rats. In rats starved 24 hours after the burn the difference of the values is probably significant (P < 0.05). It must be mentioned that the stomach of some of the control rats in this group was not quite empty, presumably due to ingestion of faeces. This fact may explain the variation in the values.

Gastric secretion In the gastric fistulae rats, a slight decrease in acid and pepsin basal secretion was noted but this decrease was not significant. TABLE II Total acid secretion

Total pepsin secretion

A

Day I

II

III IV

Basal secretion

114±36 156+53 95±42 121 ±40

Gastrin stimulated secretion

Basal secretion

280±128 271 ±60

677±229

247±107

231 ±101

884±225 499±139 793±414

Gastrin stimulated secretion

877+373 749±151 870±391 801 ±342

Gastric acid secretion collected in 10 rats, examined on 4 different days. On the days before Day II and Day III the rats were exposed to water at 370 and 80° respectively for 25 seconds. Acid secretion is expressed in ,Eq/hour and pepsin in ug/hour. 0-4 ,ug gastrin II was used as a stimulating dose.

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S. E. SVENSSON AND H. WETTERQVIST DISCUSSION

Urinary histamine The sex difference in urinary histamine excretion (Leitch, Debley and Haley, 1956; Gustafsson, Kahlson and Rosengren, 1957; Westling, 1958; Kim, 1959) is reflected in the values from burned male and female rats. When water at 60° is used, the histamine excretion tends to increase, and this is more pronounced for water at 700 and 80°. In mice (Dekanski, 1945) and dogs (Kisima, 1938) there is an increase in the skin histamine content in mice and dogs after burns, accompanied by an increase in the urinary excretion of histamine-in mice within 48 hours and in dogs within 96 hours. There is a late increase in the urinary excretion of histamine best seen in the groups dipped into water at 700 and 800. Sanyal (1962) has shown that both skin histamine and urinary histamine in the rat are increased 2-3 weeks after burning. This is also due to an increased histamine formation (cf. Wetterqvist and White, 1968). It may also be seen in burned patients (Agrup et al., 1973).

Histamine forming capacity The increased histamine forming capacity in rat skin after burns is consistent with the change in the mouse skin after burns, as shown by Schayer and Ganley (1959) and Johansson and Wetterqvist (1963). Sanyal (1962) found a decrease in the histamine content in the skin 2 and 24 hours after burns, which shows that in the first phase of injury a release of preformed histamine may be involved. Rosenthal et al. (1960), and Rocha e Silva and Rosenthal (1961) had already demonstrated that in rats there was a release of histamine from heated skin. However, they are at variance with the increased values found by Kisima (1938) and Dekanski (1945) for the skin of the mouse and the dog after burning. The reason for this is as yet unexplained. Rocha e Silva (1964), by using the drug 48/80, could release larger amounts of histamine from the hind leg in burned rats compared with control rats. In the stomach there is no significantly changed histamine formation in vitro in burned rats which are fed. However, the HFC in the stomach decreases when the rats are starved (Kahlson et al., 1964). In starved and burned rats the values are somewhat lower than in control animals (Table I). The difference is probably significant (P < 0.05) for rats starved 24 hours after the burn.

Gastric secretion The results show that in female rats subjected to a burn injury there is a tendency to reduction in acid and pepsin secretion during 18-22 hours after the burn. These findings could fit with the reduction in urinary histamine and HFC in the stomach of burned and starved female rats (Wetterqvist, unpublished). Other mechanisms might be involved as well, such as decreased mucosal blood flow. However, there is no certain increase in acid secretion 5 days after burning. At this time urinary histamine is increased to some extent (Fig. 2). There are conflicting reports in the literature concerning the effectiveness of histamine to stimulate gastric secretion in the rat. From the urinary histamine excretion in the rat after the induction of experimental burns, one may calculate that there is an overproduction of maximally 100 ,ug of histamine base per day. These figures are based on the results by Westling (1958), and Westling and

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671

Wetterqvist (1962) who studied the inactivation of histamine in the female rat. 300 ,ag of histamine base per day corresponds to 12-5 ,ug per hour. This value is below the dose considered to be the threshold value (Johansson et al., 1972). Necheles and Olsson (1942) investigated the gastric secretion in dogs after burns. They found an increased gastric secretion and acidity, but not in all the animals used. The increased volume of gastric juice seemed to depend on simultaneous infusions and on previous feeding of the dog. On the other hand, Hartman (1945) could not repeat these findings. In his dogs the free hydrochloric acid and total acid were reduced after burns. Friesen (1950), in 4 dogs subjected to burns, found a uniform but slight reduction in secretory volume with no significant alteration of gastric acidity values. It is quite clear that histamine is involved in the reaction of the rat to burn injury. The functional significance of the changes in histamine formation and excretion is still obscure. The role of histamine in increased vascular permeability in inflammation seems to be confined to early and minor events, according to Wilhelm (1962). However, in rat as well as in man (Birke et al., 1957; Tham, 1966; Agrup et al., 1973) it is quite clear that there are both early and late events where histamine may be involved. The connection of histamine formation with reparative growth shown by Kahlson et al. (for references see Kahlson and Rosengren, 1968, 1971) might be the key to the problem. REFERENCES AGRUP, P., GRANERUS, G., JACOBSSON, S., WETTERQVIST, H. & WHITE, T. (1973) Histamine Metabolism in Patients with Burn Injuries. To be published. ANGERVALL, L., BJUR6, T. & WESTLING, H. (1961). The Effect of Adrenalectomy on the Urinary Excretion of Histamine in the Rat. Acta Endocr. Kbh., 36, 467. BIRKE, G., DUNE1R, H., LiLJEDAHL, S.-0., PERNOW, B., PLANTIN, L.-O. & TROELL, L. (1957) Histamine, Catecholamines and Adrenocortical Steroids in Burns. Acta chir. scand., 114, 87. BITSCH, V. (1966) A Modification of the Hunt Method for Estimating Peptic Activity in Gastric Juice. Scand. J. clin. Lab. Invest., 18, 357. BJUR6, T., WESTLING, H. & WETTERQVIST, H. (1961) Effect of Thyroid Hormones on Histamine Formation in the Rat. Br. J. Pharmac., 17, 479. CURLING, T. B. (1842) An Acute Ulceration of the Duodenum in Cases of Burn. MedicoChir. Trans. Lond., 25, 260. DEKANSKI, J. (1945) The Effect of Cutaneous Burns on Histamine in Mice. J. Physiol. Lond., 104,151. FRIESEN, S. R. (1950) The Genesis of Gastroduodenal Ulcer Following Burns. An Experimental Study. Surgery, St Louis, 28, 123. GUSTAFSSON, B., KAHLSON, G. & RosENGREN, E. (1957) Biogenesis of Histamine Studied by its Distribution and Urinary Excretion in Germfree Rats Fed a Histaminefree Diet. Acta physiol. scand., 41, 217. HARKINS, H. N. (1938) Acute Ulcer of the Duodenum (Curling's Ulcer) as a Complication of Burns: Relation to Sepsis. Surgery, St Louis, 3, 608. HARTMAN, F. W. (1945) Curling's Ulcer in Experimental Burns. Am. J. Surg., 121, 54. HIGH, D. P., SHEPHERD, D. M. & WooDCocK, B. G. (1965) Urinary Histamine Excretion in the Gastrectomized Rat. Life Sci., 4, 787. HUNT, J. N. (1948) A Method for Estimating Peptic Activity in Gastric Contents. Biochem. J., 42, 104. JOHANSSON, J., LUNDELL, L., RoSENGREN, E. & SVENSSON, S. E. (1972) Gastric Secretion and its Facilitation as Related to Gastric Mucosal Histamine. J. Physiol. Lond., 226, 431.

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JOHANSSON, M.-B. & WETTERQVIST, H. (1963) Increased Histamine Formation in Tissues from Burned Mice. Med. exp. Basel, 8, 251. KAHLSON, G. & ROSENGREN, E. (1968) New Approaches to the Physiology of Histamine. Physiol. Rev., 48,155. KAHLSON, G. & ROSENGREN, E. (1971) Biogenesis and Physiology of Histamine. London: E. Arnold. KAHLSON, G., ROSENGREN, E. & SVENSSON, S. E. (1972) Histamine and Gastric Secretion with Special Reference to the Rat. In International Encyclopedia of Pharmacology and Therapeutics. Oxford: Pergamon Press. KAHLSON, G., ROSENGREN, E. & WESTLrNG, H. (1958) Increased Formation of Histamine in the Pregnant Rat. J. Physiol., Lond., 143, 91. KAHLSON, G., ROSENGREN, E. & THU7NBERG, R. (1963) Observations on the Inhibition of Histamine Formation. J. Physiol., Lond., 169, 467. KAHLSON, G., ROSENGREN, E., SVAHN, D. & THUNBERG, R. (1964) Mobilization and Formation of Histamine in the Gastric Mucosa as Related to Acid Secretion. J. Physiol., Lond., 174, 400. KIM, K. S. (1959) Sex Differences in Histamine Metabolism in Rats. Am. J. Physiol., 197,1258. KISIMA, H. (1938) Ueber die Bedeutung des Histamins fur Verbrennungsgifte. Fukuoka acta med., 31,49. LANE, A., Joy, A. C. & Joy, E. K. (1957) Response of the Chronic Gastric Fistula Rat to Histamine. Am. J. Physiol., 190, 221. LEITCH, J. L., DEBLEY, V. G. & HALEY, T. J. (1956) Endogenous Histamine Excretion in the Rat as Influenced by X-ray Irradiation and Compound 48/80. Am. J. Physiol., 187,307. NECHELES, H. & OLSSON, W. H. (1942) Experimental Investigation of Gastrointestinal Secretions and Motility following Burns and Their Relation to Ulcer. Surgery, St Louis, 11, 751. REUSE, J. J. (1948) Comparisons of Various Histamine Antagonists. Br. J. Pharmac., 3, 174. ROCHA E SILVA, M. (1964) Chemical Mediators of the Acute Inflammatory Reaction. Ann. N. Y. Acad. Sci., 116, 899. ROCHA E SILVA, M. & ROSENTHAL, S. R. (1961) Release of Pharmacologically Active Substances from the Rat Skin in vivo following Thermal Injury. J. Pharm. exp. Ther., 132, 110. ROSENTHA.L, S. R., HUNTER, F. R., FINEMORE, F. J. & ROMAN, I. N. (1960) On an in vivo Method of Collection of Diffusates from Skin. Thermal and Radiation Injury. Archs. int. Pharmacodyn., 126, 43. SANYAL, R. K. (1962) Histamine and 5-hydroxytryptamine Metabolism after Superficial Skin Burns. Int. Archs Allergy, 21, 326. SCHAYER, R. W. & GANLEY, 0. H. (1959) Adaptive Increase in Mammalian Histidine Decarboxylase Activity in Response to Non-specific Stress. Am. J. Physiol., 197,721. THAM, R. (1966) Liberation of Histamine in Man. Gas Chromatography of Ring Methylated Imidazole Acetic Acids in Urine. Scand. J. clin. Lab. Invest., 18, 603. WESTLING, H. (1958) The Difference in the Metabolism of Injected [14C]-histamine in Male and Female Rats. Br. J. Pharmac., 13, 498. WESTLING, H. & WETTERQVIST, H. (1962) Further Observations on the Difference in the Metabolism of Histamine in Male and Female Rats. Br. J. Pharmac., 19, 64. WETTERQVIST, H. & WHITE, T. (1968) Evaluation of Histamine Formation and Catabolism in the Rat. Scand. J. clin. Lab. Invest., 22, Suppl. 104, 13. WILHELM, D. L. (1962) The Mediation of Increased Vascular Permeability in Inflammation. Pharmac. Rev., 14, 251.