INFECTION AND IMMUNITY, Dec. 1978, p. 637-639 0019-9567/78/0022-0637$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 22, No. 3 Printed in U.S.A.
Leukocytic Endogenous Mediator in Crohn's Disease NOEL W. SOLOMONS,' CHARLES 0. ELSON,' ROBERT S. PEKAREK,2 ROBERT A. JACOB,2 HAROLD H. SANDSTEAD,2 AND IRWIN H. ROSENBERG',* Section of Gastroenterology, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637' and Human Nutrition Laboratory, Science and Education Administration, United States Department of
Agriculture, Grand Forks, North Dakota 582012 Received for publication 22 May 1978
Reduced concentrations of plasma zinc associated with elevated levels of serum leukocytic endogenous mediator activity were found in 17 patients hospitalized with acute exacerbation of Crohn's disease. Neither the decrease in plasma zinc nor the increase in leukocytic endogenous activator activity in nine outpatients with quiescent disease was significant. Leukocytic endogenous mediator activity may be increased in inflammatory conditions as well as in microbial infections. Although zinc deficiency may be common in patients with Crohn's disease, exclusive reliance on circulating zinc levels to assess zinc nutriture in active Crohn's disease may be misleading.
Crohn's disease is an idiopathic inflammatory disease that involves the large intestine (granulomatous colitis), the small intestine (regional enteritis), or both (granulomatous ileocolitis). A wide range of nutritional disorders, including zinc deficiency (7, 13, 16), have been associated with Crohn's disease. Reduced levels of circulating zinc (7, 13, 16), decreased hair zinc (16), defective taste acuity (16), and a growth response to zinc supplementation in growth-retarded adolescents (7, 13) have been reported. The concept that plasma or serum zinc levels always reflect total body zinc nutriture has been challenged by recent inquiries into the protein binding and redistribution of circulating zinc in humans. Parisi and Vallee (8) and Henkin (5) have reported that 30 to 40% of zinc in the plasma is firmly bound to a2-macroglobulins and 60 to 70% is loosely bound to albumin or amino acids. The availability of binding proteins for labile zinc (3, 14) or the affinity of the binding protein for the element (4) appear to be two major determinants of plasma zinc levels. Moreover, leukocytic endogenous mediator (LEM), a small polypeptide hormone released by granulocytes, has been implicated in the mediation of the redistribution and hepatic sequestration of zinc during stress and infections in experimental animals (6, 10); increased plasma LEM activity has also been associated with the fall in plasma zinc levels observed with infections in man (17, 18). The adult patients in our earlier report on zinc deficiency in Crohn's disease (16) were all hospitalized with an acute exacerbation of their disease at the time of study. Recently, Falchuk has reported a fairly significant reduction in 637
serum zinc levels in hospitalized patients with a wide variety of acute conditions (2). Thus, because the low plasma zinc levels in our previous studies (16) may not have been entirely attributable to the nutritional status of the patients, we examined the possible influence of LEM on circulating zinc in patients with Crohn's disease.
MATERIALS AND METHODS We studied a total of 26 patients with Crohn's disease who were enrolled in the Gastroenterology Clinic of the University of Chicago Hospitals and Clinics. The diagnosis of Crohn's disease was based on conventional clinical, radiological, and, in some cases, pathological criteria. Eight patients were male, and 18 were female; their age ranged from 14 to 60 years. Nine patients were studied during routine clinic visits when their disease was clinically quiescent, and 17 were studied shortly after admission for an acute exacerbation of their disease. In none of the hospitalized patients was any confirmation of concurrent bacterial infection documented by microbiological cultures. Nine members of the professional staff of the Gastroenterology Section (5 males and 4 females) served as healthy controls. The protocol was approved by the Human Studies Committee of the University of Chicago. All subjects gave consent after receiving a full explanation of the nature and purpose of the studies. We collected blood samples for zinc analysis with trace metal-free plastic syringes and containers, added zinc-free oxalate as an anticoagulant, and separated plasma from whole blood by centrifugation. At the same time, samples of blood were collected in glass clotting tubes. Plasma and serum samples were stored at -20'C until analysis. In patients with Crohn's disease, blood was drawn between 8 and 10 a.m.; blood was usually taken from the staff members in the early afternoon. Plasma zinc levels were determined by a modification of the method of Sinha and Gabrieli (15)
638
INFECT. IMMUN.
SOLOMONS ET AL.
respectively, of the levels in saline-injected rats (Table 1). Only the depression of zinc caused by the sera of hospitalized patients was significant (P < 0.01). When LEM activity was expressed as the mean difference in serum zinc levels between rats injected with heat-inactivated sera and rats injected with unheated filtered patient sera, only the values for patients with active Crohn's disease were significantly different (P < 0.006; Table 1). Mean plasma zinc concentrations for the subjects were 57.2 + 8.6, 64.0 + 10.7, and 71.8 ± 13 ,Ig per dl (mean ± standard deviation) for hospitalized patients, outpatients, and staff members, respectively. The mean zinc level of the hospitalized patients was significantly lower than that of the staff members (P < 0.01). For the hospitalized patients, the linear regression of plasma zinc with LEM activity (expressed as a percentage of saline control) did not significantly correlate (r = -0.242; n = 11). The mean Crohn's disease activity index score 37 for 8 outpatients and 279 ± 93 for was 71 12 hospitalized patients. This difference in score between clinically quiescent and clinically active Crohn's disease was highly significant (P < 0.001). The correlation coefficient for the regression of LEM activity (expressed as a percentage of saline control) versus Crohn's disease activity index for the hospitalized patients was significant (r = -0.627; P < 0.05).
by using a 1:5 dilution of plasma with distilled deionized water and aqueous calibration standards. Serum LEM activity was assayed by the method of Wannemacher et al. (18). Briefly, clotted samples were filtered through a membrane filter (Millipore Corp., Bedford, Mass.). Half of each sample was heated at 90'C for 30 min to inactivate any mediator present. Six rats were injected intraperitoneally with 1 ml of saline (saline controls). For each patient or healthy subject, 1 ml of unheated serum (assay) and 1 ml of heat-treated serum (heat-treated control) were injected into test animals. All rats were sacrificed 4 h later, and their serum zinc concentrations were determined. We expressed LEM activity by relating the serum zinc levels in rats injected with unheated human sera to the serum zinc levels in rats in the two control groups in the following ways: (i) as a percentage of a saline control ([zinc in rats injected with unheated patient sera/zinc in rats injected with saline] x 100); and (ii) as the difference (in micrograms per deciliter) between zinc in rats injected with heated sera and zinc in a paired rat injected with unheated sera. The National Cooperative Crohn's Disease Study Group (1) has proposed a Crohn's disease activity index based on a numerical scoring of eight factors: (i) number of stools, (ii) degree of abdominal pain, (iii) general state of well-being, (iv) extraintestinal manifestations, (v) use of Lomotil or opiates, (vi) presence of abdominal mass, (vii) degree of anemia, and (viii) weight loss. Because height data were not available to calculate ideal weight-for-height, we calculated a modified index, omitting the weight loss criterion in the 20 patients for whom complete data on the remaining seven factors were available. The Student t test was used for comparing differences between the means of groups. Simple linear regression was used for intragroup comparison of paired variables.
±
DISCUSSION The finding of significantly reduced levels of circulating zinc in acutely ill, hospitalized patients with Crohn's disease has once again been confirmed, and further insight into the mechanism has been gained. Hospitalized patients with active disease, but not outpatients with quiescent disease, showed elevated activity of LEM
RESULTS zinc levels of rats injected with
The serum filtered sera from hospitalized patients with Crohn's disease, from Crohn's outpatients, and from staff members were 76.4, 107.1, and 93.8%,
TABLE 1. LEM activity as reflected by changes in serum zinc levels of rats injected with sera from patients LEM activity
Modified Crohn's Patients
% of saline controls
Healthy controls
93.8 + 15.7b
(n =9)
Outpatients
107.1 ± 31.8
Hospitalized patients
(n =9) 76.4 ± 14.2c (n= 17)
Difference from heated controls
Plasma zinc levels
4.0 + 31.6 (n =9) 9.2 ± 62.9 (n =9) 31.4 ± 42.4d (n= 17)
71.8 ± 13.5 (n =9) 64.0 ± 10.7 (n =9) 57.2 + 8.6e (n= 12)
dexa
(Qg/dl)
See reference 1. b Results are expressed as mean ± standard deviation. c Significantly different from 100 at P < 0.01. d Significantly different from 0 at P < 0.006. e Significantly different from healthy control value at P < 0.01. f Significantly different from outpatient mean at P < 0.001.
a
disease activity in-
71 ± 37 (n =8) 279 ± 93f (n = 12)
VOL. 22, 1978
LEM IN CROHN'S DISEASE
that could account for some of the depression of plasma zinc. Our results are the first reported instance in humans of LEM elevation associated with a condition other than microbial infections and demonstrate that an inflammatory disease may activate an LEM response. The mean plasma zinc concentration of the outpatients (64.0 ± 10.7 ,ug per dl) was low in absolute terms when compared with conventional standards for normal circulating zinc levels, although it was not statistically different from control levels. Unfortunately, blood samples from the staff members were taken in the afternoon, whereas those from both patient groups were taken in the morning. A substantial postprandial decline in plasma zinc levels at 6 to 9 h after breakfast has been reported (9). This unforeseen methodological problem probably accounts for the relatively low zinc levels in the staff members and may have obscured any real difference in plasma zinc in the patients with quiescent Crohn's disease. In support of this interpretation is the fact that fasting concentrations of plasma zinc obtained between 8 and 9 a.m. from 29 men and 34 women of the Human Nutrition Laboratory staff were 85.6 ± 9.4 ,g/100 ml. There are therapeutic implications to the suggestion (7, 13, 16) that zinc nutriture is impaired in Crohn's disease. Zinc supplementation in zincdeficient patients may enhance the healing of the inflammatory lesions or reverse the growth retardation. However, acute inflammation of the bowel itself, through mediation of LEM, may contribute to depression of circulating zinc. Thus, the use of other indexes of zinc nutriture in addition to plasma concentrations of zinc would seem to be necessary for a definitive documentation of the zinc status of an individual patient with Crohn's disease. Our finding of increased plasma LEM activity in patients with active Crohn's disease also has implications for the assessment of iron and copper status. LEM has been shown to depress plasma iron levels through hepatic sequestration (12) and to elevate copper levels through stimulation of ceruloplasmin synthesis (11). Thus, the interpretation of circulating levels of these two micronutrients during the acute phases of Crohn's disease is rendered difficult. LITERATURE CITED 1. Best, W. R., J. M. Becktel, and J. W. Singleton. 1976.
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
15. 16. 17.
18.
639
Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Gastroenterology 70:439-444. Falchuk, K. H. 1977. The effect of acute disease and ACTH on serum zinc proteins. N. Engl. J. Med. 296: 1129-1137. Giroux, E. L, M. Durieux, and P. J. Schecter. 1976. A study of zinc distribution in human serum. Bioinorg. Chem. 5:211-218. Giroux, E. L., P. J. Schecter, J. Schoun, and A. Sjoerdsma. 1977. Reduced binding of added zinc in the serum of patients with decompensated hepatic cirrhosis. Eur. J. Clin. Invest. 7:71-73. Henkin, R. L. 1974. Metal-albumin-amino acid interactions: chemical and physiological interrelationship. Adv. Exp. Med. Biol. 48:299-328. Kampschmidt, R. F., H. F. Upchurch, D. L. Eddington, and L A. Pulliam. 1973. Multiple biological activities of partially purified leukocytic endogenous mediator. Am. J. Physiol. 244:530-533. McClain, C. J. 1977. Zinc deficiency: a complication of Crohn's disease. Gastroenterology 72:A76. Parisi, A. F., and B. L. Vallee. 1970. Isolation of a zinc a2-macroglobulin from human serum. Biochemistry 9: 2421-2426. Pecoud, A., P. Donzel, and J. L. Schelling. 1975. Effects of food stuffs on the absorption of zinc sulfate. Clin. Pharmacol. Ther. 17:469-474. Pekarek, R. S., and W. R. Beisel. 1971. Characterization of the endogenous mediators) of serum zinc and iron depression during infection and other stresses. Proc. Soc. Exp. Biol. Med. 138:728-731. Pekarek, R. S., M. C. Powanda, and R. W. Wannemacher. 1972. The effect of leukocytic endogenous mediator (LEM) on serum copper and ceruloplasmin concentration in the rat. Proc. Soc. Exp. Biol. Med.141: 1029-1031. Pekarek, R. S., R. W. Wannemacher, and W. R. Beisel. 1972. The effect of leukocytic endogenous mediator (LEM) on the tissue distribution of zinc and iron. Proc. Soc. Exp. Biol. Med. 140:685-688. Sandstead, H. H. 1973. Zinc nutrition in the United States. Am. J. Clin. Nutr. 26:1251-1260. Schecter, P. J., E. L Giroux, J. L Schlienger, V. Hoenig, and A. Sjoerdsma. 1976. Distribution of serum zinc between albumin and armacroglobulin in patients with decompensated hepatic cirrhosis. Eur. J. Clin. Invest. 6:147-150. Sinha, S. N., and E. R. Gabrieli. 1970. Serum copper and zinc in various pathological conditions. Am. J. Clin. Pathol. 64:570-577. Solomons, N. W., I. H. Rosenberg, H. H. Sandstead, and K. P. Vo-Khactu. 1977. Zinc deficiency in Crohn's disease. Digestion 16:87-95. Wannemacher, R. W., Jr., H. L DuPont, R. S. Pekarek, M. C. Powanda, A. Schwartz, R. B. Hornick, and W. R. Beisel. 1972. An endogenous mediator of depression of amino acids and trace metals in serum during typhoid fever. J. Infect. Dis. 126:77-86. Wannemacher, R. W., Jr., R. S. Pekarek, A. S. Klainer, P. J. Bartelloni, H. L. DuPont, R. B. Hornick, and W. R. Beisel. 1975. Detection of a leukocytic endogenous mediator-like mediator of serum amino acid and zinc depression during various infectious illnesses. Infect. Immun. 11:873-875.
Vol. 22, No. 3 Printed in U.S.A.
Leukocytic Endogenous Mediator in Crohn's Disease NOEL W. SOLOMONS,' CHARLES 0. ELSON,' ROBERT S. PEKAREK,2 ROBERT A. JACOB,2 HAROLD H. SANDSTEAD,2 AND IRWIN H. ROSENBERG',* Section of Gastroenterology, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637' and Human Nutrition Laboratory, Science and Education Administration, United States Department of
Agriculture, Grand Forks, North Dakota 582012 Received for publication 22 May 1978
Reduced concentrations of plasma zinc associated with elevated levels of serum leukocytic endogenous mediator activity were found in 17 patients hospitalized with acute exacerbation of Crohn's disease. Neither the decrease in plasma zinc nor the increase in leukocytic endogenous activator activity in nine outpatients with quiescent disease was significant. Leukocytic endogenous mediator activity may be increased in inflammatory conditions as well as in microbial infections. Although zinc deficiency may be common in patients with Crohn's disease, exclusive reliance on circulating zinc levels to assess zinc nutriture in active Crohn's disease may be misleading.
Crohn's disease is an idiopathic inflammatory disease that involves the large intestine (granulomatous colitis), the small intestine (regional enteritis), or both (granulomatous ileocolitis). A wide range of nutritional disorders, including zinc deficiency (7, 13, 16), have been associated with Crohn's disease. Reduced levels of circulating zinc (7, 13, 16), decreased hair zinc (16), defective taste acuity (16), and a growth response to zinc supplementation in growth-retarded adolescents (7, 13) have been reported. The concept that plasma or serum zinc levels always reflect total body zinc nutriture has been challenged by recent inquiries into the protein binding and redistribution of circulating zinc in humans. Parisi and Vallee (8) and Henkin (5) have reported that 30 to 40% of zinc in the plasma is firmly bound to a2-macroglobulins and 60 to 70% is loosely bound to albumin or amino acids. The availability of binding proteins for labile zinc (3, 14) or the affinity of the binding protein for the element (4) appear to be two major determinants of plasma zinc levels. Moreover, leukocytic endogenous mediator (LEM), a small polypeptide hormone released by granulocytes, has been implicated in the mediation of the redistribution and hepatic sequestration of zinc during stress and infections in experimental animals (6, 10); increased plasma LEM activity has also been associated with the fall in plasma zinc levels observed with infections in man (17, 18). The adult patients in our earlier report on zinc deficiency in Crohn's disease (16) were all hospitalized with an acute exacerbation of their disease at the time of study. Recently, Falchuk has reported a fairly significant reduction in 637
serum zinc levels in hospitalized patients with a wide variety of acute conditions (2). Thus, because the low plasma zinc levels in our previous studies (16) may not have been entirely attributable to the nutritional status of the patients, we examined the possible influence of LEM on circulating zinc in patients with Crohn's disease.
MATERIALS AND METHODS We studied a total of 26 patients with Crohn's disease who were enrolled in the Gastroenterology Clinic of the University of Chicago Hospitals and Clinics. The diagnosis of Crohn's disease was based on conventional clinical, radiological, and, in some cases, pathological criteria. Eight patients were male, and 18 were female; their age ranged from 14 to 60 years. Nine patients were studied during routine clinic visits when their disease was clinically quiescent, and 17 were studied shortly after admission for an acute exacerbation of their disease. In none of the hospitalized patients was any confirmation of concurrent bacterial infection documented by microbiological cultures. Nine members of the professional staff of the Gastroenterology Section (5 males and 4 females) served as healthy controls. The protocol was approved by the Human Studies Committee of the University of Chicago. All subjects gave consent after receiving a full explanation of the nature and purpose of the studies. We collected blood samples for zinc analysis with trace metal-free plastic syringes and containers, added zinc-free oxalate as an anticoagulant, and separated plasma from whole blood by centrifugation. At the same time, samples of blood were collected in glass clotting tubes. Plasma and serum samples were stored at -20'C until analysis. In patients with Crohn's disease, blood was drawn between 8 and 10 a.m.; blood was usually taken from the staff members in the early afternoon. Plasma zinc levels were determined by a modification of the method of Sinha and Gabrieli (15)
638
INFECT. IMMUN.
SOLOMONS ET AL.
respectively, of the levels in saline-injected rats (Table 1). Only the depression of zinc caused by the sera of hospitalized patients was significant (P < 0.01). When LEM activity was expressed as the mean difference in serum zinc levels between rats injected with heat-inactivated sera and rats injected with unheated filtered patient sera, only the values for patients with active Crohn's disease were significantly different (P < 0.006; Table 1). Mean plasma zinc concentrations for the subjects were 57.2 + 8.6, 64.0 + 10.7, and 71.8 ± 13 ,Ig per dl (mean ± standard deviation) for hospitalized patients, outpatients, and staff members, respectively. The mean zinc level of the hospitalized patients was significantly lower than that of the staff members (P < 0.01). For the hospitalized patients, the linear regression of plasma zinc with LEM activity (expressed as a percentage of saline control) did not significantly correlate (r = -0.242; n = 11). The mean Crohn's disease activity index score 37 for 8 outpatients and 279 ± 93 for was 71 12 hospitalized patients. This difference in score between clinically quiescent and clinically active Crohn's disease was highly significant (P < 0.001). The correlation coefficient for the regression of LEM activity (expressed as a percentage of saline control) versus Crohn's disease activity index for the hospitalized patients was significant (r = -0.627; P < 0.05).
by using a 1:5 dilution of plasma with distilled deionized water and aqueous calibration standards. Serum LEM activity was assayed by the method of Wannemacher et al. (18). Briefly, clotted samples were filtered through a membrane filter (Millipore Corp., Bedford, Mass.). Half of each sample was heated at 90'C for 30 min to inactivate any mediator present. Six rats were injected intraperitoneally with 1 ml of saline (saline controls). For each patient or healthy subject, 1 ml of unheated serum (assay) and 1 ml of heat-treated serum (heat-treated control) were injected into test animals. All rats were sacrificed 4 h later, and their serum zinc concentrations were determined. We expressed LEM activity by relating the serum zinc levels in rats injected with unheated human sera to the serum zinc levels in rats in the two control groups in the following ways: (i) as a percentage of a saline control ([zinc in rats injected with unheated patient sera/zinc in rats injected with saline] x 100); and (ii) as the difference (in micrograms per deciliter) between zinc in rats injected with heated sera and zinc in a paired rat injected with unheated sera. The National Cooperative Crohn's Disease Study Group (1) has proposed a Crohn's disease activity index based on a numerical scoring of eight factors: (i) number of stools, (ii) degree of abdominal pain, (iii) general state of well-being, (iv) extraintestinal manifestations, (v) use of Lomotil or opiates, (vi) presence of abdominal mass, (vii) degree of anemia, and (viii) weight loss. Because height data were not available to calculate ideal weight-for-height, we calculated a modified index, omitting the weight loss criterion in the 20 patients for whom complete data on the remaining seven factors were available. The Student t test was used for comparing differences between the means of groups. Simple linear regression was used for intragroup comparison of paired variables.
±
DISCUSSION The finding of significantly reduced levels of circulating zinc in acutely ill, hospitalized patients with Crohn's disease has once again been confirmed, and further insight into the mechanism has been gained. Hospitalized patients with active disease, but not outpatients with quiescent disease, showed elevated activity of LEM
RESULTS zinc levels of rats injected with
The serum filtered sera from hospitalized patients with Crohn's disease, from Crohn's outpatients, and from staff members were 76.4, 107.1, and 93.8%,
TABLE 1. LEM activity as reflected by changes in serum zinc levels of rats injected with sera from patients LEM activity
Modified Crohn's Patients
% of saline controls
Healthy controls
93.8 + 15.7b
(n =9)
Outpatients
107.1 ± 31.8
Hospitalized patients
(n =9) 76.4 ± 14.2c (n= 17)
Difference from heated controls
Plasma zinc levels
4.0 + 31.6 (n =9) 9.2 ± 62.9 (n =9) 31.4 ± 42.4d (n= 17)
71.8 ± 13.5 (n =9) 64.0 ± 10.7 (n =9) 57.2 + 8.6e (n= 12)
dexa
(Qg/dl)
See reference 1. b Results are expressed as mean ± standard deviation. c Significantly different from 100 at P < 0.01. d Significantly different from 0 at P < 0.006. e Significantly different from healthy control value at P < 0.01. f Significantly different from outpatient mean at P < 0.001.
a
disease activity in-
71 ± 37 (n =8) 279 ± 93f (n = 12)
VOL. 22, 1978
LEM IN CROHN'S DISEASE
that could account for some of the depression of plasma zinc. Our results are the first reported instance in humans of LEM elevation associated with a condition other than microbial infections and demonstrate that an inflammatory disease may activate an LEM response. The mean plasma zinc concentration of the outpatients (64.0 ± 10.7 ,ug per dl) was low in absolute terms when compared with conventional standards for normal circulating zinc levels, although it was not statistically different from control levels. Unfortunately, blood samples from the staff members were taken in the afternoon, whereas those from both patient groups were taken in the morning. A substantial postprandial decline in plasma zinc levels at 6 to 9 h after breakfast has been reported (9). This unforeseen methodological problem probably accounts for the relatively low zinc levels in the staff members and may have obscured any real difference in plasma zinc in the patients with quiescent Crohn's disease. In support of this interpretation is the fact that fasting concentrations of plasma zinc obtained between 8 and 9 a.m. from 29 men and 34 women of the Human Nutrition Laboratory staff were 85.6 ± 9.4 ,g/100 ml. There are therapeutic implications to the suggestion (7, 13, 16) that zinc nutriture is impaired in Crohn's disease. Zinc supplementation in zincdeficient patients may enhance the healing of the inflammatory lesions or reverse the growth retardation. However, acute inflammation of the bowel itself, through mediation of LEM, may contribute to depression of circulating zinc. Thus, the use of other indexes of zinc nutriture in addition to plasma concentrations of zinc would seem to be necessary for a definitive documentation of the zinc status of an individual patient with Crohn's disease. Our finding of increased plasma LEM activity in patients with active Crohn's disease also has implications for the assessment of iron and copper status. LEM has been shown to depress plasma iron levels through hepatic sequestration (12) and to elevate copper levels through stimulation of ceruloplasmin synthesis (11). Thus, the interpretation of circulating levels of these two micronutrients during the acute phases of Crohn's disease is rendered difficult. LITERATURE CITED 1. Best, W. R., J. M. Becktel, and J. W. Singleton. 1976.
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
15. 16. 17.
18.
639
Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Gastroenterology 70:439-444. Falchuk, K. H. 1977. The effect of acute disease and ACTH on serum zinc proteins. N. Engl. J. Med. 296: 1129-1137. Giroux, E. L, M. Durieux, and P. J. Schecter. 1976. A study of zinc distribution in human serum. Bioinorg. Chem. 5:211-218. Giroux, E. L., P. J. Schecter, J. Schoun, and A. Sjoerdsma. 1977. Reduced binding of added zinc in the serum of patients with decompensated hepatic cirrhosis. Eur. J. Clin. Invest. 7:71-73. Henkin, R. L. 1974. Metal-albumin-amino acid interactions: chemical and physiological interrelationship. Adv. Exp. Med. Biol. 48:299-328. Kampschmidt, R. F., H. F. Upchurch, D. L. Eddington, and L A. Pulliam. 1973. Multiple biological activities of partially purified leukocytic endogenous mediator. Am. J. Physiol. 244:530-533. McClain, C. J. 1977. Zinc deficiency: a complication of Crohn's disease. Gastroenterology 72:A76. Parisi, A. F., and B. L. Vallee. 1970. Isolation of a zinc a2-macroglobulin from human serum. Biochemistry 9: 2421-2426. Pecoud, A., P. Donzel, and J. L. Schelling. 1975. Effects of food stuffs on the absorption of zinc sulfate. Clin. Pharmacol. Ther. 17:469-474. Pekarek, R. S., and W. R. Beisel. 1971. Characterization of the endogenous mediators) of serum zinc and iron depression during infection and other stresses. Proc. Soc. Exp. Biol. Med. 138:728-731. Pekarek, R. S., M. C. Powanda, and R. W. Wannemacher. 1972. The effect of leukocytic endogenous mediator (LEM) on serum copper and ceruloplasmin concentration in the rat. Proc. Soc. Exp. Biol. Med.141: 1029-1031. Pekarek, R. S., R. W. Wannemacher, and W. R. Beisel. 1972. The effect of leukocytic endogenous mediator (LEM) on the tissue distribution of zinc and iron. Proc. Soc. Exp. Biol. Med. 140:685-688. Sandstead, H. H. 1973. Zinc nutrition in the United States. Am. J. Clin. Nutr. 26:1251-1260. Schecter, P. J., E. L Giroux, J. L Schlienger, V. Hoenig, and A. Sjoerdsma. 1976. Distribution of serum zinc between albumin and armacroglobulin in patients with decompensated hepatic cirrhosis. Eur. J. Clin. Invest. 6:147-150. Sinha, S. N., and E. R. Gabrieli. 1970. Serum copper and zinc in various pathological conditions. Am. J. Clin. Pathol. 64:570-577. Solomons, N. W., I. H. Rosenberg, H. H. Sandstead, and K. P. Vo-Khactu. 1977. Zinc deficiency in Crohn's disease. Digestion 16:87-95. Wannemacher, R. W., Jr., H. L DuPont, R. S. Pekarek, M. C. Powanda, A. Schwartz, R. B. Hornick, and W. R. Beisel. 1972. An endogenous mediator of depression of amino acids and trace metals in serum during typhoid fever. J. Infect. Dis. 126:77-86. Wannemacher, R. W., Jr., R. S. Pekarek, A. S. Klainer, P. J. Bartelloni, H. L. DuPont, R. B. Hornick, and W. R. Beisel. 1975. Detection of a leukocytic endogenous mediator-like mediator of serum amino acid and zinc depression during various infectious illnesses. Infect. Immun. 11:873-875.
