Biochemical detection of fecal hematin as a test for meat eating in chimpanzees (Pan troglodytes).код для вставкиСкачать
American Journal of Primatology 3:327-332 (1982) TECHNICAL NOTE Biochemical Detection of Fecal Hematin as a Test for Meat Eating in Chimpanzees (Pan troglodytes) FRANK SPENCER1, NOEL T BOAZ', MEL ALLEN3, AND W C McGREW4 Deporfnwnt of Anthropology, Queens C o l l r g ~of the City linwerszty of New York, Fludzing. New York, 'Department of Anthropology, New York [Jniuerstty, New York Clty, Neu1 York, 3Departrnent ofAnthropology, [Jnzwrsity of Oklahoma, Norman, Oklahomq and 4D~partment of Psychology, University of Stirzing, Stirling, Scotland Meat-eating by wild chimpanzees has been reported by a number of workers during the last two decades. Direct observation probably underestimates the incidence, even though the behavior occurs relatively infrequently. In 1978, Moreno-Black suggested that fecal analysis over a long period of time is probably the most effective means to determine the incidence of this behavior in wild chimpanzees and other nonhuman primate groups. A method currently employed by a number of fieldworkers involves the recovery of the remains of a carnivorous meal in the animal's feces. This method, however, may also under represent the incidence because of 1)complete digestion of mammalian parts, 2) the unidentifiability of partially digested remains, and 3) the reingestion of feces. This paper reports the results of a laboratory study using a fecal test not subject t o these limitations. The test is based on the biochemical detection of hematin, a derivative of hemaglobin which is found in all mammalian tissues. The results of this study reveal that hematin is a reliable indicator of meat consumption. The test is available in a commercially prepared kit, namely HEMOCULT, which was developed to detect clinically significant amounts of blood in the feces of human patients with presumptive gastrointestinal lesions. This kit has been evaluated with a view to its possible application in the field. Key words: Chimpanzee, Pun, meat eating, fecal analysis INTRODUCTION Within the last two decades field studies of wild chimpanzees have revealed that meat comprises a small but consistent part of the diet of the species [Goodall, 1963; Hladik, 1977; McGrew, in press; McGrew e t al., 1978,1979: Nishida e t al., 1979; Suzuki, 1975; Teleki, 1973; Wrangham, 19751. Direct observation probably greatly underestimates the incidence of meat eating, although this type of behavior occurs relatively rarely. Moreno-Black  has suggested that fecal analysis over a long period is the most effective means to determine the importance of meat eating in a chimpanzee group's diet. McGrew et al.  reported results of analyses over a 2 year period of 380 fecal samples from Mt. Assirik, Senegal, in which 2.6% of the specimens contained signs of meat eating. This method, which relies on the recognition of the remains of a carnivorous meal, may also under represent the incidence of meat eating because of 1)complete Received November 24, 1981; accepted March 12, 1982. Address reprint requests to Dr. Frank Spencer, Department of Anthropology, Queens College of the City University of New York, Kissend Boulevard, Flushing, New York 11367. 0275-2565/82/0301-04-~327$02.00 0 1982 Alan R. Liss, Inc. 328 Spencer et a1 digestion of some animal parts, 2) the unidentifiability of partially digested remains [Harding, 19811 and 3) the occasional reingestion of feces [Wrangham, 1975; McGrew et d., 19791. We report here a fecal test not subject to these limitations, based on the biochemical detection of hematin, a derivative of hemoglobin. The test is available in a commercially packaged kit, namely HEMOCULT'", which was developed to detect clinically significant amounts of occult blood in the feces of human subjects with presumptive gastrointestinal lesions. This kit has been evaluated with a view to its possible application in the field. Presuming that chimpanzees in nature do not normally eal meat daily and that the amounts consumed vary from meal to meal depending on social and environmental factors, we designed the following simple experiment. I t seeks to simulate natural conditions and to determine: 1)if differing amounts of meat in a diet are reflected in varying amounts of hematin in the feces; 2) if the HEMOCULT'" test can detect these predicted changes in the concentration of hematin; 3) if the gut-passage time, namely the time elapsed from the ingestion of the meat to the appearance of hematin in the feces, can be measured; and 4) if the sensitivity of the HEMOCULT" test is affected by the age of the specimen. Throughout the study, the reliability and sensitivity of the HEMOCULT'" test was monitored by standard laboratory in vitro procedures. METHODS A group of communally living chimpanzees (3 males and 3 females) at the Institute for Primate Studies of the University of Oklahoma was used in this study (Table I). This group was considered ideal because they were accustomed to eating meat, and known not to be coprophagous. Over an 11-dayperiod, this group received a modified Radcliffe diet (Table 11) to which was added on prescribed days an increasing amount of uncooked, ground beef. The living quarters of the group were routinely cleaned twice a day, namely morning and afternoon, which permitted the collection of fecal samples the approximate time of deposition of which were known. The morning specimens were collected between 0800 and 1000 hours; the afternoon samples were collected between 1500 and 1700 hours. From days 1 through 3 the chimpanzees received a Radcliffe meat-free diet. Fecal samples collected during this period provided a baseline of meat-free consumption. On day 4 each animal received approximately 93 g (0.25lb) of uncooked meat mixed in their diet. On day 5 the subjects were returned to a meat-free diet, which was maintaineduntil day 8, whereupon they received approximately 187 g (0.5 lb) of uncooked meat. For the remaining 3 days of the study the animals were returned to a meat-free diet. Fecal samples were sealed in 10 ml glass, screw-capped vials and stored at 4°C until analyzed (see below). In addition to the HEMOCULT'" test, the collected fecal samples were analyzed for hematin using standard laboratory in vitro techniques. Such tests for hematin depend upon the fact that heme proteins catalyze the oxidation of organic substances such as benzidine and orthotolidine by hydrogen peroxide. The reaction produces a blue color, the intensity of which is mostly a function of the concentration of hematin. The reaction is like that catalyzed by true peroxidases, but the tests are made specific by boiling the feces. This destroys peroxidases of bacterial or vegetable origin, leaving only the heme compounds, which are thermostable. In Vitro Techniques Reagents: 1)Saturated solution of either benzidine or orthotolidine. Dissolve 1.0 g of benzidine dihydrochloride in distilled water and dilute to 100 ml. Mix, filter, and store in a dark bottle at 4°C. To prepare the orthotolidine solution, dissolve 1.08 g of orthotolidine in absolute methanol, and dilute to 100 ml. Store in a dark bottle at 4°C. 2) 3% solution of hydrogen peroxide. Test for Meat Eating in Chimpanzees 329 TABLE I. Chimpanzees Tested, Institute for Primale Studies, University of Oklahoma* Name Sex McCarthy Nim Onan Kelly Lilly Sherrv M M M F F F Weight (kg) 36 32 42 39 40 31 Date of Birth Origin c. 12- 7-71 11-19-73 5-25-72 C. 9-23-72 C. 1- 1-72 C. 3-14-74 wild captivity captivity wild wild wild *All females were nulliparous, although Lilly was approximately 7 months pregnant during the study. TABLE 11. Individual Daily Diet* Constituents Amounts Carrots Isonicotinic acid hydrozyde‘ Vitamin B6 Ascorbic acid Bean sprouts Grain mix2 Water 150 g 300 mg 100 mg 6g 60 g 275 g 750 mg *Modified Radcliffe diet. ‘Tuberculosis prophylactic. ’Constituents: maize corn, wheat, barley, oats, soybean meal, alfalfa meal, yeast, and ground oyster shell. NOTE: The usnal diet, except for the purpose of this study contains approximately 45 g of cooked meat (skimmed of fat) for each of the individuals used as subjects. Procedure: 1)Thoroughly mix fecal specimen using a wooden applicator stick, and then transfer a portion the size of a large pea to the bottom of each of 2 graduated 15 ml centrifuge tubes. Label 1 of these tubes: “positive control.” To this tube add 0.05 ml of (human) blood. Add 5 ml of distilled water to each tube and mix thoroughly. Add distilled water to the 10 ml mark and again mix thoroughly. 2) Centrifuge tubes at approximately 700 X g for 10 minutes. 3) Transfer 1 ml supernatant from each tube into an appropriately labeled test tube. 4) To a third test tube labeled “negative control,” add 1 ml of distilled water. 5) Boil all three test tubes for 5 minutes. 6) Transfer test tubes to a beaker containing water at room temperature. 7 ) When test tubes have cooled, add to each 1 ml of benzidine or orthotolidine solution, followed immediately by 1 ml of hydrogen peroxide solution. 8) After 1 minute examine each test tube for color. If the test is positive, the experimental tube will be distinctly blue like the positive control tube. The negative control tube should be colorless. Because benzidine and orthotolidine are very sensitive and prone to give false-positive results, one must run controls with each batch of tests. Furthermore, all positive results should be replicated. All tests in this study were done twice, and all with positive results were repeated by another worker. Using serial dilutions of a standard aqueous solution of crystalline human hemoglobin mixed with human feces, the above tests were estimated to be sensitive to approximately 3 mg hemoglobin/ gm of feces. HEMOCULT Test Kit: The HEMOCULT’” it is manufactured by SmithKline Diagnostics, PO Box 61947, Sunnyvale, CA 94086. I t consists of a specially prepared guaiac- 330 Spencer et a1 impregnated filter paper housed in a specially designed envelope which eases identification, preparation, and subsequent development of the test. The test utilizes the principle that hemoglobin exerts a peroxidase-like activity and promotes the oxidation of the guaiac resin by hydrogen peroxide. A positive result is signaled by the appearance of a blue color on or at the edge of the smear. RESULTS AND DISCUSSION Figure 1 shows that the results of the in vitro tests and the HEMOCULT'" slide test gave high agreement. Because the HEMOCULT'" system does not remove bacterial or vegetable peroxidases, we expected problems with false positives. This seems to have been unfounded. We later found that clinical trials had been done, and all reported the HEMOCULT" system to be virtually free of false positives, even with specimens from patients on unrestricted diets [Ostrow et al., 1973; Fleisher et al., 1977; Glober and Peskoe, 1974; Winawer et al., 19761. This evidently has been achieved by reducing the sensitivity of the test, which is clearly less accurate than the standard in vitro procedures (Ostrow et al., 19731. The latter authors found that the HEMOCULT" system is sensitive to a minimum of 5 mg of hemoglobin/gm of feces. At this level of concentration the test gives equivocal results. However, with more than 10 mg of hemoglobin/ gm of feces, the results are reliable [Ostrow et al., 1973: 9331. As for the possibility of false positives due to gastrointestinal bleeding, all of the subjects used in our study were known to be in good health. Also all of the obtained positive results (Fig. I) were confined to specimens where the presence of occult blood was expected. Because in the field an investigator may not know the physical condition of the animal whose fecal sample is being assessed, the presence of a sick animal in a group could confound an investigation. But while recognizing that the HEMOCULT'" system is not sensitive to the origin of blood in the feces, it is suggested that this inherent dif- HB4OCULT TOLIDINE ; 0 BENZIDINE + -+ n 1 0 L Fig. 1. Detection of hematin by three methods (top)compared with presence of meat in diet (bottom).0 = negative result; ? = equivocal result; = positive result. + Test for Meat Eating in Chimpanzees 331 ficulty may be overcome in a number of ways. For instance, the recognition of a pathological specimen: such as 1)the presence of frank blood, which generally indicates a rectal or anal lesion, or 2) encountering a black-colored (melaena) stool which has a tarlike consistency and invariably indicates bleeding in the upper gastrointestinal tract. Besides the macroscopic appearance of the feces, the investigator’s suspicion should also be alerted by the continuing presence of a single positive stool over a period of time in a group in which all other specimens deposited in the same period are negative. I t is common knowledge among field workers that chimpanzees regularly defecate several times daily. In the wild the chimpanzee is primarily a frugivore, consuming large amounts of indigestible material such as pits and seeds of fruit. Hence, it seems reasonable to suggest that the frequency of bowel movements is largely a physiological response to the accumulation of this material and the residue of digestion in the lower intestinal tract. In this regard, Milton has noted that frugivorousprimates have a much faster gut-passage time than do folivorous-herbivorous species -a difference that might explain the relatively fast gut-passage time of chimpanzees compared to humans. However, the extent to which this apparently rapid passage of food through the alimentary canal affects the ability of the chimpanzee to deal effectively with the digestion of complex animal proteins is not known. This question has important implications for hominid evolution and requires further investigation. From our limited study, the gutpassage time in the Oklahoma group of chimpanzees was found to be between 6 and 24 hours, which is presumed not to be significantly different from that of chimpanzees in nature. Furthermore, a microscopic examination of the fecal specimens revealed the presence of meat fibres in two of the three specimens with a positive occult blood test. Also, the general consistency of the stools from days 4 through 11 were soft and loose compared with those collected on days 1 through 3, which were firmer and more formed. The significance of these observations remains to be seen. In order to determine the effect of a specimen’s age on results, the specimens which had proved positive were stored at room temperature and tested over 3 weeks at intervals of 7 days. Though they still tested positively after 2 weeks, the color was fainter. After 3 weeks the results were equivocal. These results largely confirm the findings of Ostrow et al. [1973: 9351, but Fleisher et al. showed that a strong-positive reaction will occur for up to 30 days with specimens applied to the guaiac impregnated filter papers. For weakly testing positives the HEMOCULT‘“ test is reportedly unstable, and sometimes after only 4 or 5 days tests become negative. This suggests that our initial results were of intermediate strength. The results obtained under the prescribed conditions of this study seem to show that the I-IEMOCULT’” system reliably detects hematin, but several questions need to be answered before the test can be used routinely in the field. First, it is not known if the normal diet of wild chimpanzees would affect the specificity of the test. For example, there is evidence that ascorbic acid in the diet might distort the results. According to Jaffe [I9781 ingesting more than 250 mg of ascorbic acid may cause false-negative results. Evidently the amount in the diet of the subjects of this study did not do so. Also, it is not known how the kit will perform in a tropical climate. The manufacturer recommends that the kits be stored at “controlled room temperature 15-30°C (59-86°F)”with “protect[ionl from heat and light.” These questions, however, can only be answered by trials in the field. CONCLUSION This study indicates that hematin in the feces of chimpanzees is a reliable indicator of meat consumption which can be detected by relatively simple biochemical procedures. Although the present study could not evaluate the HEMOCULT” slide test under natural conditions, the results nevertheless indicate that it should be tried in the field as a quick and easy method of detecting meat eating. I t might also be useful in studies of other omnivorous species [Harding, 19811. 332 Spencer et a1 ACKNOWLEDGMENTS This study was supported by a Queens College of the City University of New York Faculty Research Grant. The authors are indebted to Dr. C. E. Hopla, Department of Zoology, and Dr. W. B. Lemmon, Director, Institute for Primate Studies, University of Oklahoma, Norman, for their assistance. Also, Andrew Sillen for initial discussions which led to the development of this approach. REFERENCES Fleisher, M.; Schwartz, M.K.; Winawer, S.J. Laboratory studies on the HEMOCULT'" slide for fecal occult blood, pp. 181-187 in COLORRECTAL CANCER PREVENTION, EPIDEMIOLOGY AND SCREENING. S. Winawer and D. Schottenfeld, eds. New York, Raven Press, 1980. Glober, G.A.; Peskoe, S.M. Outpatient screening for gastrointestinal lesions using guaiacimpregnated slides. AMERICAN JOURNAL OF DIGESTIVE DISORDERS 19:399-403, 1974. Goodall, J. Feeding behavior of wild chimpanzees: A prehminary report. SYMPOSIA OF T H E ZOOLOGICAL SOCIETY OF LONDON 10139-47,1963. Harding, R.S.O. An order of omnivores: Nonhuman primate diets in the wild, pp. 191-214 in OMNIVOROUS PRIMATES. R.S.O. Harding and G. Teleki, eds. New York, Columbia University Press, 1981. Hladik, C.M. Chimpanzees of Gabon and Chimpanzees of Gombe: Some comparative data on t h e diet, pp. 481-501 in PRIMATE ECOLOGY. T.H. Clutton-Brock, ed. New York, Academic Press, 1977. Jaffe, R.M. False-negative stool occult blood tests caused by ingestion of ascorbic acid (Vitamin C). ANNALS OF INTERNAL MEDICINE 832324,1975. McGrew, W.C. Animal foods in the diets of wild chimpanzees: Why cross-cultural variation? CARNIVORE, in press. McGrew, W.C.; Tutin, C.E.G.; Baldwin, P.J.; Sharman, M.J.; Whiten, A. Primates preying upon vertebrates: New records from West Africa. CARNIVORE 1:41-45, 1978. McGrew, W.C.; Tutin, C.E.G.; Baldwin, P.J. New data on meat-eating by wild chimpanzees. CURRENT ANTHROPOLOGY 20: 238-239,1979. Milton, K. Food choice and digestive strategies of two sympatric primate species. AMERICAN NATURALIST 117:496-503,1981, Moreno-Black, G. The use of scat samples in primate diet analysis. PRIMATES 19: 215-222,1978. Nishida, T.; Uehara, S.; Nyundo, R. Predatory behavior among wild chimpanzees of the Mahali Mountains. PRIMATES 2O:l-20, 1979. Ostrow, J.D.; Mulvaney, C.A.; Hansell, J.R.; Rhodes, R.S. Sensitivity and reproducibility of chemical tests for fecal occult blood with emphasis on false-positive reactions. AMERICAN JOURNAL OF DIGESTIVE DISEASES 18:930-940, 1973. Suzuki, A. The origin of hominid hunting: A primatological perspective, pp. 259-278 in SOCIOECOLOGY AND PSYCHOLOGY OF PRIMATES. R.H. Tuttle, ed. The Hague, Mouton, 1975. Teleki, G. THE PREDATORY BEHAVIOR O F WILD CHIMPANZEES. Lewisburg, Bucknell University Press, 1973. Winawer, S.J.; Sherlock, P.; Schottenfeld, D.; Miller, D.G. Screening for colon cancer. GASTROENTEROLOGY 70:783-789, 1976. Wrangham, R. The behavioral ecology of chimpanzees in Gombe National Park, Tanzania. Ph.D. Dissertation, University of Cambridge, 1975.