Serum Lysozyme, Serum Proteins, and Immunoglobulin Determinations in Nonspecific Inflammatory Bowel Disease O.S. EL-KHATIB, MD, O. L E B W O H L , MD, A.A. ATTIA, MD, C.A. FLOOD, MD, J.A. STEIN, MD, J.G. SWEETING, MD, R.T. WHITLOCK, MD, E.F. OSSERMAN, MD, and P.R. HOLT, MD
The serum levels of lysozyme, serum electrophoresis, and serum immunoglobulins were determined prospectively in 101 patients with ulcerative colitis, ulcerative proctitis, Crohn 's disease, or nonclassifiable nonspecific inflammatory bowel disease. Although the mean serum lysozyme concentration of patients with Crohn's disease (10.5 +_6.8 tzg/ml) and ulcerative colitis (9.6 + 4.1 lzg/ml) performed by a standardized lysoplate method was significantly greater than normal controls (6.0 +- 1.5 tzg/ml), the results did not correlate with the diagnosis nor with the degree of disease activity. Individually separated protein fractions and serum immunoglobulins also did not correlate with the serum lysozyme levels. This study indicates that measurement of the level o f serum lysozyme in individual patients is not helpful in determining the cause or degree of activity o f nonspecific inflammatory bowel disease.
The differential diagnosis of nonspecific inflammatory bowel disease into ulcerative colitis, Crohn's disease, and ulcerative proctitis has preoccupied a major portion of the recent literature on these disorders. The observations by Falchuk et al (1) suggesting that Crohn's disease and ulcerative colitis could be distinguished by measurement of the serum level of lysozyme (muramidase) promised to end the confusion. These authors' initial observations were challenged by several investigators (25). Nugent et al (6) demonstrated a significant increase in the mean serum lysozyme concentration From the Departments of Medicine and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York. This study was supported by National Cancer Institute Grant CA 02332 and NIH Training Grant AM 05499 and Research Grant AM 13436. Address for reprint requests: Dr. P. R. Holt, St. Luke's Hospital Center, 114 St. and Amsterdam Avenue, New York, New York 10025.
in patients with Crohn's disease and Peeters et al (7) found significant elevations in some patients with active Crohn's disease. Subsequently, Falchuk and associates reported that serum lysozyme might not be elevated in Crohn's disease (8) of mild to moderate activity and was occasionally found increased in ulcerative colitis (9). The present study was undertaken shortly after the initial report of Falchuk et al and was designed to c o m p a r e serum l y s o z y m e c o n c e n t r a t i o n s in patients with differing forms of inflammatory bowel disease and assess disease activity at the time that the blood was drawn without knowledge of the resuits of the enzyme determinations. In addition to the lysozyme assays, protein electrophoresis and immunoglobulin quantitation by immunodiffusion were performed on the same serum samples. A total of 101 patients were carefully evaluated before the results of the laboratory determinations were reviewed.
Digestive Diseases, Vol. 23, No. 4 (April 1978)
0002-9211/78/0400-0297505.09/19 1978DigestiveDisease Systems, Inc.
297
EL-KHATIB E T AL
Fig 1. S e r u m
lysozyme data from 101 patients divided into diag-
categories as described in the text. Shaded area designates normal range. nostic
MATERIALS AND METHODS
Serum was obtained in consecutive patients with inflammatory bowel disease who had a blood urea nitrogen determination of less than 20 mg/100 ml serum and from whom informed consent was obtained. The serum was kept refrigerated or frozen, and a single serum lysozyme
determination was performed within 10 days using the lysoplate method (10) with human lysozyme purified from the urine of monocyte leukemic subjects by the bentonite procedure (10) as the reference standard. The extinction coefficient (E2s0 rim) of the lysozyme prepared by this method is 2.46. By this method, the normal values for serum lysozyme based upon 37 laboratory and professional personnel aged 20-50 years is 6.0 -+ 1.5 (so), range 3.7-10 ~g/ml. Serum protein electrophoresis was performed on cellulose acetate strips (Microzone| method, Beckman Instrument Co.). Immunoglobulins were quantitated by single radial immunodiffusion using Immunoplates| (Kallestad Corp.). The diagnosis of Crohn's disease was based upon unequivocal demonstration of noncaseating granulomas frequently associated with other accepted pathologic criteria (11) (21 patients), radiologic evidence of small-intestinal involvement (12) (20 patients), or radiological changes considered characteristic of Crohn's disease involving the large intestine, eg, skip areas, fistulas, and strictures (12) (14 patients). The diagnosis of ulcerative colitis was based upon contiguous involvement of the co!on judged by radiologic arid endoscopic examination, absence of radiologic evidence of small-intestinal disease, histologic criteria characteristic of ulcerative colitis on large-bowel biopsies, and evidence of involvement above 35 cm from the anus. Ulcerative proctitis was diagnosed in patients with uniform disease, restricted to the rectum or to the colon up to 35 cm from the anus for a minimum of 6 months, histologic criteria similar to those for ulcerative colitis, and absence of systemic symptoms. Five patients with chronic nonspecific inflammatory bowel disease but who did not strictly fulfill the above criteria were termed "unclassified." At the time serum was drawn, the patients' disease was categorized as active or inactive. In ulcerative colitis and ulcerative proctitis the activity was based upon symptoms, fever, leukocytosis, and sedimentation rate by criteria similar to those described by T r u e l o v e and Witts (13). Activity in Crohn's disease was judged by the presence of fever, elevated sedimentation rate, leukocytosis or increased immature granulocytes, and changes in clinical symptoms when compared to a previously wellestablished baseline.
TABLE 1. SERUM PROTEIN ELECTROPHORESIS
Protein (gMI) Disease category Ulcerative colitis Ulcerative proctitis Crohn's
disease Normal range
Number of patients*
Albumin Mean +- SD (range)
al Mean • SD (range)
e~2 M e a n + SD (range)
fl Mean • so (range)
Yl M e a n +_ SD (range)
Yz M e a n • SD (range)
23
3.41 • 0.59 (2.10-4.30) 3.81 • 0.32 (3.10-4.30) 3.36 -+ 0.62
0.37 -+ 0.10 (0.30-0.70) 0.33 • 0.07 (0.20-0.50) 0.37 • 0.07
0.86 • 0.16 (0.60-1.40) 0.85 • 0.18 (0.60-1.40) 0.93 _ 0.17
0.84 -4- 0.15 (0.60-1.20) 0.81 --_ 0.13 (0.60-1.40) 0.90 • 0.14
0.30 • 0.08 (0.20-0.50) 0.34 • 0.06 (0.20-0.40) 0.46 • 0.34
1.33 • 0.48 (0.60-3.20) 1.14 • 0.25 (0.70-1.60) 1.18 • 0.30
(1.70-4.60) (3.50-4.80)
(0.20-0.50) (0.40-0.80)
(0.60-1.30) (0.50-0,90)
(0.60-3.90) (0.70-1.20)
(0.10-0:90) ( 0-0.30)
(0.60-1.90) (0.90-1.80)
17 55
*The number of determinations may be less than the number of patients described in Materials and Methods section if insufficient serum was available for analysis.
298
Digestive Diseases, Vol. 23, No. 4 (April 1978)
LYSOZYME IN INFLAMMATORY BOWEL DISEASE RESULTS Elevated serum lysozyme concentrations (over 10 /~g/ml) were found in 35% of 55 patients with Crohn's disease, 38% of 23 patients with ulcerative colitis, 17% of 18 patients with ulcerative proctitis, and 2 of 5 unclassified patients. The mean -+ sD of the serum l y s o z y m e d e t e r m i n a t i o n s was 10.50 +_ 6.8 in Crohn's disease, 9.64 _+ 4.1 in ulcerative colitis, significantly higher than normal (P < 0.05), and 8.51 --- 2.5 in ulcerative proctitis and 10.76 ___ 3.4 in the unclassified patients (not significantly differing from the normals). The results of single lysozyme determinations in the four diagnostic categories are shown in Figure 1. When serum lysozyme levels of patients with active and inactive disease were compared (Figure 2), no differences were found nor were differences with activity apparent within any diagnostic group. Neither the extent of bowel involvement nor treatment affected the results. Table 1 presents the results of the electrosphoretic analyzed serum proteins and Table 2 the quantitative immunoglobin data. No significant differences in any group of patients were found nor were differences detected when cases were subdivided into degrees of activity. The level of serum lysozyme was not statistically correlated with the results of any of the serum proteins or any specific immunoglobulin. DISCUSSION Our data are in agreement with previous studies suggesting that the mean serum lysozyme level in ulcerative colitis and Crohn's disease is higher than the mean of normal subjects (5, 6), but they indicate that the concentration in individual patients
Fig 2. S e r u m l y s o z y m e data from 101 patients w h o s e disease activity w a s characterized as active or inactive. Shaded area designates normal range.
is no help in differential diagnosis. The variable result.s found in these and other studies (14, 15) have been ascribed to differences in performing lysozyme assays (16, 17). Differences in methodology are unlikely to be responsible for the discrepancies between the results of our studies and those of Falchuk and associates since they also used the lyso-
TABLE 2. SERUM IMMUNOGLOBULINS
Immunoglobu!ins (g/dl) Disease category Ulcerative colitis Ulcerative proctitis Crohn's disease Normal range
Number of patients*
IgG Mean -2--SD (range)
IgA Mean +- SD (range)
IgM Mean • SD (range)
IgD Mean • sD (range)
22
1260 • 473 (500-2800) 1103 • 372 (600-1800) 1091 • 415 (350-2300) (700-1700)
238 4- 98 (64-440) 191 • 106 (36-400) 245 • 135 (36-640) (100-350)
174 4- 104 (60-600) 208 • 98 (86-400) 211 • 97 (60-620) (50-200)
0.45 • 0.9 (0-1) 1.0 • 1.5 (0-2) 1.28 • 1.8 (0-7) . (0-10)
18 51
*The n u m b e r of determinations m a y be less than the n u m b e r of patients described in Materials and M e t h o d s section if insufficient s e r u m was available for analysis.
Digestive Diseases, Vol. 23, No. 4 (April 1978)
299
EL-KHATIB
plate method with purified human lysozyme standards obtained from our laboratory. The mean and range of serum lysozyme values in normal subjects in our laboratory was lower than that reported by Falchuk et al, and most of the elevated levels of serum lysozyme in their study were over 20/xg/ml. Only 2 of our patients (both with Crohn's disease) had lysozyme determinations above 20/~g/ml. The hypothesis which prompted the studies of Falchuk and associates was that disorders in which tissue granuloma formation was characteristic, eg, tuberculosis and sarcoidosis, are accompani.ed by elevated serum lysozyme levels (reviewed in ref. 1). Monocytes which continuously release lysozyme are the precursors of macrophages, epitheloid cells, and giant cells, all components of tissue granulomas. Although the presence of noncaseating granulomas is characteristic of Crohn's disease, they have not been found in surgical specimens of 30-70% of patients in whom other accepted histologic features, such as transmural involvement, were present (18). Presumably, if lysozyme appearing in excess in the serum were to be derived from active granulomata, the level would be expected to relate to the extent of involvement and number of granulomas present in diseased tissue. In that regard, it is of interest that the serum lysozyme was normal in three of our patients who underwent subtotal colectomy and in whom the bowel was extensively involved with active granulomas. The specimen of one patient with a serum lysozyme above 15 ~g/ml on several occasions was found to have only 10 cm of involved small intestine showing no histologic evidence of granuloma formation. The serum of mice in which granulomas were induced in the footpad by injections of Crohn's disease tissue also failed to show e l e v a t e d l y s o z y m e concentrations (19). No significant changes in the mean concentration of the serum protein electrophoretic fractions were noted in any diagnostic category of non-specific inflammatory disease of the bowel, deDombal (20) has previously reported that severe ulcerative colitis resulting in early surgery is accompanied by a low or falling level of gamma globulin and elevated a-2-globulin. In the present studies 12% of patients with ulcerative colitis had low levels of gamma globulin, but this finding was not associated with a poor prognosis. Furthermore, quantitative determinations of the serum immunoglobulins by the Mancini immunodiffusion technique failed to discriminate between diagnostic categories or to correlate
300
ET AL
with increased severity of the disease. These data are contrary to a report which suggested that serum immunoglobulins were normal in ulcerative proctitis but that serum IgG and IgA levels were elevated in ulcerative colitis (21). Finally, the serum lysozyme concentration did not correlate with any serum protein fraction, This study fails to demonstrate diagnostic discrimination for the measurement of serum lysozyme in inflammatory bowel disease or to relate the level of disease activity. We must therefore conclude the serum lysozyme determination is of no help in the management of patients with these disorders. However, the meaning of greatly elevated levels in the sera of some patients remains an intriguing observation that requires further experimental studies. Intestinal lysozyme is concentrated in Paneth cells of the small intestine, and the enzyme is discharged into the lumen by some pharmacologic stimuli or under certain physiologic conditions (22). In addition, immunoglobulins, particularly IgA are found in small-intestinal Paneth cells (23). The possibility that patients with inflammatory bowel disease who have considerable elevations of serum lysozyme might have altered Paneth cell function appears worth pursuing. ACKNOWLEDGMENT
We gratefully acknowledge the assistance of Mrs. J. Shyong and MsB. Jones. REFERENCES 1. Falchuk KR, Perrotto JL, Isselbacher K J: Serum lysozyme in Crohn's disease and ulcerative colitis. N Engl J Med 292:395-397, 1975 2. Pounder RE, Avella JR, McCallum H, Misiewicz J J: Serum lysozyme in inflammatory bowel disease. Lancet 2:228-229, 1975 (letter) 3. Pruzanski W, Marcon N: Lysozyme in Crohn's disease. N Engl J Med 293:611-612, 1975 (letter) 4. Kane SP, Hoffbrand AV, Meale G: Indices of granulocyte activity in inflammatory bowel disease. Gut 15:953 959, 1974 5. Hylander E, Hansen NE, Karle H, Janrum S: Serum lysozyme in Crohn's disease and ulcerative colitis. Scand J Gastroenterol 11:2t3-217, 1976 6. Nugent FE, Mallari R, George H, Ridley N: Serum lysozyme in inflammatory bowel disease. Gastroenterology 70:1014-1016, 1976 7. Peeters TL, Van Trappen G, Geboes K: Serum lysozyme levels in Crohn's disease and ulcerative colitis. Gut 17:300305, 1976 8. Falchuk KR, Perrotto JL, Isselbacher KJ: Serum lysozyme in Crohn's disease. A useful index of disease activity. Gastroenterology 69:893-896, 1975 Digestive Diseases, Vol. 23, No. 4 (April 1978)
LYSOZYME IN INFLAMMATORY
BOWEL DISEASE
9. Falchuk KR, Perrotto JL, Isselbacher KJ: Serum lysozyme levels in Crohn's disease. N Engl J Med 292:1350, 1975 (letter) 10. Osserman EF, Lawlor DP: Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med 124:921-952, 1966 11. Lockhart-Mummery HG, Morson BC: Crohn's disease of the large intestine and its distinction from ulcerative colitis. Gut 1:87-105, 1960 12. Bernard S, Wolf BS, Marshak RH: Granulomatous colitis (Crohn's disease of the colon). Am J Roentgenol Radium Ther Nucl Med 88:662-670, 1962 13. Truelove SC, Witts LJ." Cortisone in ulcerative colitis: Final report on a therapeutic trial. Br Med J 2:1041-1044, 1955 14. Belaiche J, Vesin P, Cattan D, Zittoun J, Bernier JJ: Serum lysozyme in inflammatory bowel disease. Gastroenterology 70:633, 1976 (letter) 15. Schussheim A, Josephson AS, Greenwald RA: Serum lyso-
Digestive Diseases, Vol. 23, No. 4 (April1978)
16. 17. 18.
19.
20. 21.
22.
zyme in inflammatory bowel disease. Gastroenterology 70:632-633, 1976 (letter) Johansson BG, Ursing B: Lysozyme in Crohn's disease: Influence of methods. N Engl J Med 294:337, 1976 (letter) Isselbacher K: Serum lysozyme in inflammatory bowel disease. Gastroenterology 70:634, 1976 (letter) Schachter H, Kirsner JB: Definitions of inflammatory bowel disease of unknown etiology. Gastroenterology 68:591-598, 1975 Borak E, Taub R, EI-Khatib O, Osserman E, Holt PR: Serum lysozyme in mice with induced granulomas. N Engl J Med 295:623, 1976 (letter) deDombal FT: Prognostic value of serum proteins during severe attacks of ulcerative colitis. Gut 9:144-149, 1968 Smith HJ, Macphee IW: A clinico-immunological study of ulcerative colitis and ulcerative proctitis. Gut 12:20-26, 1971 Peeters TL, Van Trappen G: The Paneth cell: A source of intestinal lysozyme. Gut 16:553-558, 1975
30 1
The serum levels of lysozyme, serum electrophoresis, and serum immunoglobulins were determined prospectively in 101 patients with ulcerative colitis, ulcerative proctitis, Crohn 's disease, or nonclassifiable nonspecific inflammatory bowel disease. Although the mean serum lysozyme concentration of patients with Crohn's disease (10.5 +_6.8 tzg/ml) and ulcerative colitis (9.6 + 4.1 lzg/ml) performed by a standardized lysoplate method was significantly greater than normal controls (6.0 +- 1.5 tzg/ml), the results did not correlate with the diagnosis nor with the degree of disease activity. Individually separated protein fractions and serum immunoglobulins also did not correlate with the serum lysozyme levels. This study indicates that measurement of the level o f serum lysozyme in individual patients is not helpful in determining the cause or degree of activity o f nonspecific inflammatory bowel disease.
The differential diagnosis of nonspecific inflammatory bowel disease into ulcerative colitis, Crohn's disease, and ulcerative proctitis has preoccupied a major portion of the recent literature on these disorders. The observations by Falchuk et al (1) suggesting that Crohn's disease and ulcerative colitis could be distinguished by measurement of the serum level of lysozyme (muramidase) promised to end the confusion. These authors' initial observations were challenged by several investigators (25). Nugent et al (6) demonstrated a significant increase in the mean serum lysozyme concentration From the Departments of Medicine and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York. This study was supported by National Cancer Institute Grant CA 02332 and NIH Training Grant AM 05499 and Research Grant AM 13436. Address for reprint requests: Dr. P. R. Holt, St. Luke's Hospital Center, 114 St. and Amsterdam Avenue, New York, New York 10025.
in patients with Crohn's disease and Peeters et al (7) found significant elevations in some patients with active Crohn's disease. Subsequently, Falchuk and associates reported that serum lysozyme might not be elevated in Crohn's disease (8) of mild to moderate activity and was occasionally found increased in ulcerative colitis (9). The present study was undertaken shortly after the initial report of Falchuk et al and was designed to c o m p a r e serum l y s o z y m e c o n c e n t r a t i o n s in patients with differing forms of inflammatory bowel disease and assess disease activity at the time that the blood was drawn without knowledge of the resuits of the enzyme determinations. In addition to the lysozyme assays, protein electrophoresis and immunoglobulin quantitation by immunodiffusion were performed on the same serum samples. A total of 101 patients were carefully evaluated before the results of the laboratory determinations were reviewed.
Digestive Diseases, Vol. 23, No. 4 (April 1978)
0002-9211/78/0400-0297505.09/19 1978DigestiveDisease Systems, Inc.
297
EL-KHATIB E T AL
Fig 1. S e r u m
lysozyme data from 101 patients divided into diag-
categories as described in the text. Shaded area designates normal range. nostic
MATERIALS AND METHODS
Serum was obtained in consecutive patients with inflammatory bowel disease who had a blood urea nitrogen determination of less than 20 mg/100 ml serum and from whom informed consent was obtained. The serum was kept refrigerated or frozen, and a single serum lysozyme
determination was performed within 10 days using the lysoplate method (10) with human lysozyme purified from the urine of monocyte leukemic subjects by the bentonite procedure (10) as the reference standard. The extinction coefficient (E2s0 rim) of the lysozyme prepared by this method is 2.46. By this method, the normal values for serum lysozyme based upon 37 laboratory and professional personnel aged 20-50 years is 6.0 -+ 1.5 (so), range 3.7-10 ~g/ml. Serum protein electrophoresis was performed on cellulose acetate strips (Microzone| method, Beckman Instrument Co.). Immunoglobulins were quantitated by single radial immunodiffusion using Immunoplates| (Kallestad Corp.). The diagnosis of Crohn's disease was based upon unequivocal demonstration of noncaseating granulomas frequently associated with other accepted pathologic criteria (11) (21 patients), radiologic evidence of small-intestinal involvement (12) (20 patients), or radiological changes considered characteristic of Crohn's disease involving the large intestine, eg, skip areas, fistulas, and strictures (12) (14 patients). The diagnosis of ulcerative colitis was based upon contiguous involvement of the co!on judged by radiologic arid endoscopic examination, absence of radiologic evidence of small-intestinal disease, histologic criteria characteristic of ulcerative colitis on large-bowel biopsies, and evidence of involvement above 35 cm from the anus. Ulcerative proctitis was diagnosed in patients with uniform disease, restricted to the rectum or to the colon up to 35 cm from the anus for a minimum of 6 months, histologic criteria similar to those for ulcerative colitis, and absence of systemic symptoms. Five patients with chronic nonspecific inflammatory bowel disease but who did not strictly fulfill the above criteria were termed "unclassified." At the time serum was drawn, the patients' disease was categorized as active or inactive. In ulcerative colitis and ulcerative proctitis the activity was based upon symptoms, fever, leukocytosis, and sedimentation rate by criteria similar to those described by T r u e l o v e and Witts (13). Activity in Crohn's disease was judged by the presence of fever, elevated sedimentation rate, leukocytosis or increased immature granulocytes, and changes in clinical symptoms when compared to a previously wellestablished baseline.
TABLE 1. SERUM PROTEIN ELECTROPHORESIS
Protein (gMI) Disease category Ulcerative colitis Ulcerative proctitis Crohn's
disease Normal range
Number of patients*
Albumin Mean +- SD (range)
al Mean • SD (range)
e~2 M e a n + SD (range)
fl Mean • so (range)
Yl M e a n +_ SD (range)
Yz M e a n • SD (range)
23
3.41 • 0.59 (2.10-4.30) 3.81 • 0.32 (3.10-4.30) 3.36 -+ 0.62
0.37 -+ 0.10 (0.30-0.70) 0.33 • 0.07 (0.20-0.50) 0.37 • 0.07
0.86 • 0.16 (0.60-1.40) 0.85 • 0.18 (0.60-1.40) 0.93 _ 0.17
0.84 -4- 0.15 (0.60-1.20) 0.81 --_ 0.13 (0.60-1.40) 0.90 • 0.14
0.30 • 0.08 (0.20-0.50) 0.34 • 0.06 (0.20-0.40) 0.46 • 0.34
1.33 • 0.48 (0.60-3.20) 1.14 • 0.25 (0.70-1.60) 1.18 • 0.30
(1.70-4.60) (3.50-4.80)
(0.20-0.50) (0.40-0.80)
(0.60-1.30) (0.50-0,90)
(0.60-3.90) (0.70-1.20)
(0.10-0:90) ( 0-0.30)
(0.60-1.90) (0.90-1.80)
17 55
*The number of determinations may be less than the number of patients described in Materials and Methods section if insufficient serum was available for analysis.
298
Digestive Diseases, Vol. 23, No. 4 (April 1978)
LYSOZYME IN INFLAMMATORY BOWEL DISEASE RESULTS Elevated serum lysozyme concentrations (over 10 /~g/ml) were found in 35% of 55 patients with Crohn's disease, 38% of 23 patients with ulcerative colitis, 17% of 18 patients with ulcerative proctitis, and 2 of 5 unclassified patients. The mean -+ sD of the serum l y s o z y m e d e t e r m i n a t i o n s was 10.50 +_ 6.8 in Crohn's disease, 9.64 _+ 4.1 in ulcerative colitis, significantly higher than normal (P < 0.05), and 8.51 --- 2.5 in ulcerative proctitis and 10.76 ___ 3.4 in the unclassified patients (not significantly differing from the normals). The results of single lysozyme determinations in the four diagnostic categories are shown in Figure 1. When serum lysozyme levels of patients with active and inactive disease were compared (Figure 2), no differences were found nor were differences with activity apparent within any diagnostic group. Neither the extent of bowel involvement nor treatment affected the results. Table 1 presents the results of the electrosphoretic analyzed serum proteins and Table 2 the quantitative immunoglobin data. No significant differences in any group of patients were found nor were differences detected when cases were subdivided into degrees of activity. The level of serum lysozyme was not statistically correlated with the results of any of the serum proteins or any specific immunoglobulin. DISCUSSION Our data are in agreement with previous studies suggesting that the mean serum lysozyme level in ulcerative colitis and Crohn's disease is higher than the mean of normal subjects (5, 6), but they indicate that the concentration in individual patients
Fig 2. S e r u m l y s o z y m e data from 101 patients w h o s e disease activity w a s characterized as active or inactive. Shaded area designates normal range.
is no help in differential diagnosis. The variable result.s found in these and other studies (14, 15) have been ascribed to differences in performing lysozyme assays (16, 17). Differences in methodology are unlikely to be responsible for the discrepancies between the results of our studies and those of Falchuk and associates since they also used the lyso-
TABLE 2. SERUM IMMUNOGLOBULINS
Immunoglobu!ins (g/dl) Disease category Ulcerative colitis Ulcerative proctitis Crohn's disease Normal range
Number of patients*
IgG Mean -2--SD (range)
IgA Mean +- SD (range)
IgM Mean • SD (range)
IgD Mean • sD (range)
22
1260 • 473 (500-2800) 1103 • 372 (600-1800) 1091 • 415 (350-2300) (700-1700)
238 4- 98 (64-440) 191 • 106 (36-400) 245 • 135 (36-640) (100-350)
174 4- 104 (60-600) 208 • 98 (86-400) 211 • 97 (60-620) (50-200)
0.45 • 0.9 (0-1) 1.0 • 1.5 (0-2) 1.28 • 1.8 (0-7) . (0-10)
18 51
*The n u m b e r of determinations m a y be less than the n u m b e r of patients described in Materials and M e t h o d s section if insufficient s e r u m was available for analysis.
Digestive Diseases, Vol. 23, No. 4 (April 1978)
299
EL-KHATIB
plate method with purified human lysozyme standards obtained from our laboratory. The mean and range of serum lysozyme values in normal subjects in our laboratory was lower than that reported by Falchuk et al, and most of the elevated levels of serum lysozyme in their study were over 20/xg/ml. Only 2 of our patients (both with Crohn's disease) had lysozyme determinations above 20/~g/ml. The hypothesis which prompted the studies of Falchuk and associates was that disorders in which tissue granuloma formation was characteristic, eg, tuberculosis and sarcoidosis, are accompani.ed by elevated serum lysozyme levels (reviewed in ref. 1). Monocytes which continuously release lysozyme are the precursors of macrophages, epitheloid cells, and giant cells, all components of tissue granulomas. Although the presence of noncaseating granulomas is characteristic of Crohn's disease, they have not been found in surgical specimens of 30-70% of patients in whom other accepted histologic features, such as transmural involvement, were present (18). Presumably, if lysozyme appearing in excess in the serum were to be derived from active granulomata, the level would be expected to relate to the extent of involvement and number of granulomas present in diseased tissue. In that regard, it is of interest that the serum lysozyme was normal in three of our patients who underwent subtotal colectomy and in whom the bowel was extensively involved with active granulomas. The specimen of one patient with a serum lysozyme above 15 ~g/ml on several occasions was found to have only 10 cm of involved small intestine showing no histologic evidence of granuloma formation. The serum of mice in which granulomas were induced in the footpad by injections of Crohn's disease tissue also failed to show e l e v a t e d l y s o z y m e concentrations (19). No significant changes in the mean concentration of the serum protein electrophoretic fractions were noted in any diagnostic category of non-specific inflammatory disease of the bowel, deDombal (20) has previously reported that severe ulcerative colitis resulting in early surgery is accompanied by a low or falling level of gamma globulin and elevated a-2-globulin. In the present studies 12% of patients with ulcerative colitis had low levels of gamma globulin, but this finding was not associated with a poor prognosis. Furthermore, quantitative determinations of the serum immunoglobulins by the Mancini immunodiffusion technique failed to discriminate between diagnostic categories or to correlate
300
ET AL
with increased severity of the disease. These data are contrary to a report which suggested that serum immunoglobulins were normal in ulcerative proctitis but that serum IgG and IgA levels were elevated in ulcerative colitis (21). Finally, the serum lysozyme concentration did not correlate with any serum protein fraction, This study fails to demonstrate diagnostic discrimination for the measurement of serum lysozyme in inflammatory bowel disease or to relate the level of disease activity. We must therefore conclude the serum lysozyme determination is of no help in the management of patients with these disorders. However, the meaning of greatly elevated levels in the sera of some patients remains an intriguing observation that requires further experimental studies. Intestinal lysozyme is concentrated in Paneth cells of the small intestine, and the enzyme is discharged into the lumen by some pharmacologic stimuli or under certain physiologic conditions (22). In addition, immunoglobulins, particularly IgA are found in small-intestinal Paneth cells (23). The possibility that patients with inflammatory bowel disease who have considerable elevations of serum lysozyme might have altered Paneth cell function appears worth pursuing. ACKNOWLEDGMENT
We gratefully acknowledge the assistance of Mrs. J. Shyong and MsB. Jones. REFERENCES 1. Falchuk KR, Perrotto JL, Isselbacher K J: Serum lysozyme in Crohn's disease and ulcerative colitis. N Engl J Med 292:395-397, 1975 2. Pounder RE, Avella JR, McCallum H, Misiewicz J J: Serum lysozyme in inflammatory bowel disease. Lancet 2:228-229, 1975 (letter) 3. Pruzanski W, Marcon N: Lysozyme in Crohn's disease. N Engl J Med 293:611-612, 1975 (letter) 4. Kane SP, Hoffbrand AV, Meale G: Indices of granulocyte activity in inflammatory bowel disease. Gut 15:953 959, 1974 5. Hylander E, Hansen NE, Karle H, Janrum S: Serum lysozyme in Crohn's disease and ulcerative colitis. Scand J Gastroenterol 11:2t3-217, 1976 6. Nugent FE, Mallari R, George H, Ridley N: Serum lysozyme in inflammatory bowel disease. Gastroenterology 70:1014-1016, 1976 7. Peeters TL, Van Trappen G, Geboes K: Serum lysozyme levels in Crohn's disease and ulcerative colitis. Gut 17:300305, 1976 8. Falchuk KR, Perrotto JL, Isselbacher KJ: Serum lysozyme in Crohn's disease. A useful index of disease activity. Gastroenterology 69:893-896, 1975 Digestive Diseases, Vol. 23, No. 4 (April 1978)
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