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Fecal Lactate and Ulcerative Colitis
GASTROENTEROLOGY 1988;95:1564-8
Fecal Lactate and Ulcerative Colitis PIERO VERNIA, RENZO CAPRILLI, GIOVANNI LATELLA, FABRIZIO BARBETTI, FABIO M. MAGLIOCCA, and MAURO CITTADINI
Department of Gastroenterology, Universita "La Sapienza," Rome, and Department of Gastroenterology, Universita de L'Aquila, L'Aquila, Italy
Impaired metabolism of short-chain fatty acids, as well as a modified fecal ionogram, have been reported in ulcerative colitis. Fecal water samples from 62 patients with ulcerative colitis were analyzed in the present investigation to evaluate changes in SCFAs and lactic acid in relation to activity and severity of disease. Short-chain fatty acid levels were high in quiescent and mild disease (162.6 ± 63.6 and 147.8 ± 63.2 mMlL, respectively), but significantly decreased in the severe form (64.7 ± 46.9 mM/L). Lactate showed a progressive increase from mild colitis (3.0 ± 1.8 mM/L) to severe colitis (21.4 ± 18.6 mM/L). It thus appears that mild colitis displayed a fecal pattern characterized by normal pH and bicarbonate, slightly impaired electrolyte handling, high short-chain fatty acid values, and only moderately increased lactate. Severe colitis, on the other hand, was characterized by low fecal pH, bicarbonate, and potassium, high sodium and chloride, low short-chain fatty acid levels, and very high lactate levels. A critical lowering of intraluminal pH, which shifts bacterial metabolism from shortchain fatty acid to lactate production, may be responsible for the intraluminal pooling of lactate.
S
hort-chain fatty acids (SCFAs), products of bacterial fermentation of unabsorbed carbohydrates, represent a primary energy source for colonic mucosal cells (1). Impaired absorption or utilization of SCFAs, or both, by the colonic mucosa has recently been described in patients with ulcerative colitis (UC) (2,3), suggesting a change in colonocyte metabolism. In preliminary studies we reported an abnormal fecal organic ionogram in UC, mainly characterized by an increase in lactate (4). The fecal excretion of bicarbonate is reduced and fecal pH is low in patients with active UC (5,6). Interestingly, in experimental studies on rats, the association of high intrduminal content of lactate (100 mM/L) and low
pH «4) reduced water and electrolyte absorption and induced colonic mucosal damage (7). Furthermore, in our laboratory mucosal alterations strictly resembling those of human UC were induced in rats by the infusion of lactic acid at a concentration of 25 mM/L, which is of the same order as those found in the stools of patients with UC (8). It seems, therefore, that the change in organic anion metabolism, associated with low intraluminal pH, could be involved in the onset or deterioration of colitic lesions. The aim of the present study was, therefore, to evaluate the changes in fecal pH, SCFAs, and lactate in relation to the activity and severity of colitis.
Patients and Methods Patients The study was carried out on 62 patients with UC (5 quiescent, 57 active disease) admitted to the GI Unit of the University of Rome during the period 1982-86, and 29 healthy controls. Diagnosis was based on clinical, radiologic, endoscopic, and histologic findings (9). The severity of the disease was assessed according to the criteria of Truelove and Witts (10). Colitis was classified as severe in 20, moderate in 23, and mild in"14 patients. The extent of the lesions was established from radiologic (doublecontrast barium enema), endoscopic (total colonoscopy), and histologic findings. Twenty-four patients whose lesions spread beyond the transverse colon were considered to have total colitis. The lesions did not reach the midtransverse colon in the 19 cases classified as left-sided colitis. Thirteen patients had proctosigmoiditis. Data from patients with quiescent colitis were evaluated separately. No patient was taking oral corticosteroids or using local corticosteroids before the stool collection.
Abbreviutions used in this puper: SCF A, short-chain fatty acid; UC, ulcerative colitis. © 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
FECAL ORGANIC ANIONS IN DC
December 1988
1565
Table 1. Fecal Wet Weight, pH, and Electrolytes in Relation to the Severity of Colitis Colitis
Fecal weight pH Na+ K+ ClCa 2+ Mg2+ K+/Na+
Controls
Quiescent
Mild
Moderate
Severe
(29)
(5)
(14)
(23)
(20)
149.2 6.8 18.2 61.2 13.8 26.0 41.6 6.9
± ± ± ± ± ± ± ±
101.3 0.7 17.1 25.1 10.8 18.8 33.2 6.1
234.9 7.2 32.5 52.5 28.3 27.6 28.0 2.7
± ± ± ± ± ± ± ±
221.1 6.4 30.4 51.6 23.1 44.4 53.3 3.1
123 0.7 14.9 18.8 18.3 26.0 22.4 3.1
± ± ± ± ± ± ± ±
82.7 d
346.3 6.3 47.6 39.8 36.3 30.8 25.6 1.1
0.8 20.2 d 22.0 14.8 d 29.6 42.8 3.1c
± ± ± ± ± ± ± ±
165.4" 0.5 b 23.5" 13.7 b 17.9" 22.0 7.6 c 0.8"
± ± ± ± ± ± ± ±
541.0 6.2 62.7 35.7 50.4 16.8 14.0 0.6
418.9" O.4 b 22.8" 17.9" 23.9" 10.4 d 12.4 b 0.5"
Data expressed in mM/L; fecal weight in grams per 24 h. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.0001, b P < 0.001, C P < 0.01, d P < 0.05 versus controls.
Fecal Analysis Twenty-four-hour stool specimens were collected in preweighed, air-tight plastic cans upon admission. Thiomersal (15 ml, 1: 10,000 solution) was added immediately after the first evacuation to minimize bacterial overgrowth. Fecal water was extracted using an in vitro dialysis method and analyzed immediately or frozen and stored at a temperature of -20°C (11). Organic anions were analyzed by gas-liquid chromatography (12), and their concentration was calculated against that of standard solutions of the following acids: acetic, propionic, isobutyrie, butyric, isovaleric, valerie, isocaproie, and caproic. Concentrations of L- and D-lactic acid were measured by an enzymatic method using specific L- and D-lactate dehydrogenase (Boehringer Biochem) (13). Sodium, potassium, calcium, and magnesium concentrations were measured by flame photometry and chloride by potentiometric titration. The stool pH was measured on freshly passed stools by a glass electrode connected to a Beckman pH-meter (Beckman Instruments, Fullerton, Calif.).
Statistical Analysis Data were analyzed by Student's t-test for unpaired data. Correlation analysis was performed in normal controls and in patients with UC, considering all patients as a single population, and in each individual subgroup. Despite the fact that only correlations showing a p value
< 0.001 were taken into account, many variables were found to be significantly correlated. A multivariate analysis (Forward Multiple Linear Regression Test) was thus performed, considering as dependent variables the 24-h fecal wet weight, fecal pH, and concentration and output of sodium, potassium, chloride, lactic acid, and SCFAs. Only those parameters significantly influencing the dependent variable (p < 0.001) were taken into consideration.
Results The fecal wet weight and pH and inorganic component of fecal water are shown in Tables 1 and 2. Stool weight and sodium and chloride concentrations increased with the severity and extent of the disease, whereas the potassium level, potassium to sodium ratio, and pH showed an opposite trend. Similar modifications were also found in patients with quiescent disease. Concentrations of the organic anions of fecal water are shown in Tables 3 and 4. All SCF As were found to be slightly increased in quiescent and mild colitis, almost normal in moderate disease, but significantly reduced in severe colitis. Lactic acid concentration, on the other hand, showed an extremely significant increase in all patients with colitis. The increase of lactic acid was mainly due to the L-form of the acid (Figure 1). This
Table 2. Fecal Wet Weight, pH, and Electrolytes in Relation to the Extent of the Lesions
Fecal weight pH Na K ClCa 2+ Mg2+ K+/Na+
Controls
Proctosigmoiditis
Left-sided colitis
Total colitis
(29)
(13)
(20)
(24)
149.2 6.8 18.2 61.2 13.8 26.0 43.6 6.9
± ± ± ± ± ± ± ±
101.3 0.7 17.1 25.1 10.8 18.8 33.2 6.1
234.9 6.1 32.5 52.5 28.3 28.8 32.4 2.7
± ± ± ± ± ± ± ±
123.2" O.4 b 14.9 c 18.4 18.3 c 32.4 23.6 3.1 b
327.6 5.9 46.4 37.2 38.5 24.4 46.4 1.1
± ± ± ± ± ± ± ±
132.4° 0.5 b 23.1" 15.9" 21.8 0 12.4 48.8 0.7 0
510.7 6.5 59.4 38.5 42.9 33.6 31.2 1.1
± ± ± ± ± ± ± ±
398" 0.4 26.8" 18.10 23.2" 26.4 26.8 1.3"
Data expressed in mM/L; fecal weight in grams per 24 h. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.0001, b P < 0.001, C P < 0.01 versus controls.
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VERNIA ET AL.
GASTROENTEROLOGY Vol. 95, No.6
Table 3. Organic Anions in Relation to the Severity of the Colitis Colitis Controls (29) Acids Acetic Propionic Isobutyric Butyric Isovaleric Valeric Isocaproic Caproic Total SCFAs L-Lactic D-Lactic Total lactic
71.5 17.3 1.9 13.7 2.4 2.3 0.8 0.3 111.4
± ± ± ± ± ± ± ± ±
Quiescent (5)
47.3 8.4 1.0 8.4 1.8 1.2 0.2 0.4 58.6
0.2 ± 0.1 0.3 ± 0.2 0.5 ± 0.2
113.4 23.3 2.4 16.3 3.1 2.7 0.03 0.2 162.6
± ± ± ± ± ± ± ± ±
Mild (14)
60.7 10.5 1.1 4.0 1.8 2.0 0.04 0.1 63.6
96.3 28.2 2.1 17.7 2.5 2.5 0.1 0.3 147.8
± ± ± ± ± ± ± ± ±
Moderate (23)
36.6 14.8 d 1.9 11.1 2.6 3.9 0.2 0.5 63.2
63.9 22.9 1.3 14.6 1.4 1.7 0.7 0.1 106.0
2.0 ± 1.6 b 1.0 ± 1.2 3.0 ± 1.80
0.6 ± 0.8 0.9 ± 1.5 1.5 ± 2.4
± ± ± ± ± ± ± ± ±
35.7 30.0 0.8 d 13.2 0.9 d 1.5 0.2 0.2 74.1
7.8 ± 7.20 1.1 ± 1.5 8.9 ± 7.0 0
Severe (20) 47.8 10.2 0.9 4.3 1.2 0.6 0.3 0.07 64.7
± ± ± ± ± ± ± ± ±
32.1 d 8.7 b 1.3 b 5.0 0 1.7 d 0.7 0 1.0 O.ld 46.9 b
20.1 ± 13.3 0 1.3 ± 11.3 C 21.4 ± 18.60
SCFAs, short-chain fatty acids. Data expressed in mMIL. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.001, b P < 0.005, C P < 0.01, d P < 0.02 versus controls.
finding was also present in patients with quiescent colitis, although values were not statistically significant.
Correlation Analysis in Normal Subjects Linear regression analysis. Fecal weight showed a positive linear correlation with sodium (r = 0.62) and chloride (r = 0.54), but a negative correlation with fecal pH (r = -0.47). Multiple linear regression analysis. Fecal wet weight was significantly influenced only by sodium concentration and fecal pH. Sodium output was related to that of butyric acid, potassium, chloride, and lactic acid.
Correlation Analysis in Patients With Ulcerative Colitis Linear regression analysis. The 24-h fecal wet weight showed good correlation values with the concentration of sodium (r = 0.48) and the output of sodium (r = 0.95), chloride (r = 0.90), potassium (r = 0.69), lactic acid (r = 0.58), and SCFAs (r = 0.51). The concentrations of SCFAs showed negative correlations with sodium (r = -0.54) and chloride concentrations (r = -0.51), but a positive correlation with that of potassium (r = 0.42). Positive correlations were also found between lactic acid output and fecal weight (r = 0.58), and the output of sodium (r = 0.58), chloride (r = 0.49), and acetic acid (r = 0.42). Multiple linear regression. Once the independent variable "sodium" was taken into account, no
Table 4. Organic Anions in Relation to the Extent of the Lesions Controls (29) Acids Acetic Propionic Isobutyric Butyric Isovaleric Valeric Isocaproic Caproic Total SCFAs L-Lactic D-Lactic Total lactic
71.5 17.3 1.9 13.7 2.4 2.3 0.8 0.3 111.4
± ± ± ± ± ± ± ± ±
47.3 8.4 1.0 8.4 1.8 1.2 0.2 0.4 58.6
0.2 ± 0.1 0.3 ± 0.2 0.5 ± 0.2
Proctosigmoiditis (13) 81.3 31.0 1.6 15.3 2.1 2.4 0.2 0.3 129.8
± ± ± ± ± ± ± ± ±
44.4 39.0 1.6 14.9 2.3 3.1 0.5 0.6 98.4
8.1 ± 12.3 c 1.4 ± 1.1 9.5 ± 10.9 b
Left-sided colitis (20) 67.8 16.5 1.3 11.9 1.6 1.3 0.3 0.08 101.4
± ± ± ± ± ± ± ± ±
37.3 13.0 1.1 d 9.7 1.6 l.4 b 1.1 O.ld 56.1
9.6 ± 10.8 b 1.1 ± 1.4 c 10.7 ± 10.3 0
Total colitis (24) 56.8 16.1 1.3 9.6 1.5 1.2 0.4 0.1 86.8
± ± ± ± ± ± ± ± ±
35.9 12.0 1.5 11.2 1.7 d 2.2 d 0.2 0.2 58.3
12.9 ± 12.9 b 1.2 ± 1.7C 14.1 ± 17.80
SCFAs, short-chain fatty acids. Data expressed in mMIL. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.001, b P < 0.01, C P < 0.02, d P < 0.05 versus controls.
FECAL ORGANIC ANIONS IN UC
December 1988
•
' - -_ _....II D-LACTIC ACID
25
wn;:m
L-LACTIC ACID
• p<
0.0001
20
15
10
5
o
.
:::::::::
T :::::::::
r----L---;:::::::::: ......... ................. ........ ........ ........ .. ........ ................. ........ ........ . .................. :::::::: :::::::::: T :::::::: :::::::::: .....--'---1;.. .. .. •• .. . . . . . . . . .................. ............... .. r--_TL--t.:;~~ ••••••••••••••••• •••••• , ••• ........................... ......... ........ -:::::::::
.
CONTROLS QUIESCENT COLITIS
MILO COLITIS
,,"",-rn-rnM/•••••••••
MODERATE COLITIS
SEVERE COLITIS
Figure 1. Lactic acid concentration in the stools of patients with ulcerative colitis and normal controls. Mean values (mM/L) ± SEM (vertical bars). Probability values express differences versus controls.
other variable significantly influenced stool weight. The 24-h losses of sodium, on the other hand, were related with decreasing statistical weight to chloride output, stool weight, and potassium and lactic acid output. Chloride losses were shown to be influenced mainly by the output of sodium, but also by the output of lactic acid and butyric acid. Besides all individual SCFAs, only fecal pH was shown to influence SCFA concentration, whereas the concentration of lactic acid varied in relation to those of sodium and butyric acid. Output of lactic acid was again influenced by that of sodium and butyric acid, but also by that of chloride and acetic acid.
Discussion As already reported in previous investigations (14,15), the stools of patients with active UC are characterized by high 24-h weight, high concentrations of sodium and chloride, low concentrations of potassium, reduced potassium to sodium ratio, and low pH. These changes progressively increase with the severity of the disease and extent of the lesions. Similar changes are also present in patients with
1567
quiescent colitis, with the exception of pH, which is normal. As far as the organic component is concerned, fecal SCFAs are high in patients with mild disease, do not differ from controls in moderate disease, and are significantly low in severe cases. Lactate, which is present in negligible concentrations in the fecal water of normal subjects and patients with quiescent colitis, was found to be significantly increased in patients with active disease, reaching very high concentrations in severe colitis. The high concentration of SCFAs observed in mild colitis may be attributed to their impaired utilization by the colonic mucosa as suggested by Roediger (2,3). The decreased amount of coenzyme A in the mucosa of patients with UC reported some years ago (16) may account for the reduced oxidation of SCFAs (3). This hypothesis is also supported by the close similarity existing between colonic lesions in UC and those reported in experimental pantothenic acid deficiency colitis in pigs (17). It is well known that coenzyme A intervenes at different levels of intracellular metabolic pathways, i.e., l3-oxidation of SCFAs and the Krebs' cycle. Its deficit, therefore, results in an impaired utilization of SCFAs, which are the main energetic fuel of the colonic mucosa, with consequent impairment of all intracellular energydependent processes such as electrolyte exchanges and mucus and structural protein synthesis. Other factors, such as increased production of SCFAs due to less effective absorption of dietary carbohydrates (18) or diminished SCFA absorption by the inflamed mucosa (19), cannot be ruled out. In our series of patients with severe colitis, however, the fecal concentration of SCF As was significantly lower than in normal controls, suggesting that factors other than impaired SCF A absorption and utilization are present. It is noteworthy that the reduction of SCFAs was paralleled by increasing lactate concentrations and lowering of pH. The low fecal pH, due to reduced colonic bicarbonate secretion (5,6), likely reflects the low intraluminal pH. An acidic intraluminal environment is unfavorable for some SCFA-producing strains but not for lactateproducing strains, and shifts bacterial metabolic pathways from SCFA to lactate production (20-24). Other mechanisms may also lead to increased lactate production in colitis. The increased intraluminal oxygen concentration, resulting from profuse bleeding, favors facultative anaerobic strains such as lactobacilli and streptococci (24), which are lactic acid producers. Finally, the increased mucus production, the disruption of the colonic mucosal cell lining, and the exposure of mesenchymal polysaccharides (hyaluronic acid) to intraluminal bacteria may also lead to lactate production (24).
1568
VERNIA ET AL.
On the basis of stool analysis, two different phases may be recognized in the clinical course of uc. Mild colitis displays a fecal pattern characterized by normal fecal pH, normal excretion of bicarbonate, high concentrations of SCFAs, and only mildly increased lactate. Severe colitis is characterized by low fecal pH, reduced excretion of bicarbonate, low concentrations of SCFAs, and markedly increased lactic acid. The critical lowering of bicarbonate secretion and intracolonic pH may be responsible for the altered fecal water composition found in severe colitis. Low pH in fact reduces the availability of SCFAs, and at the same time enhances the production of lactic acid. This in turn may contribute to colonic damage through a direct mechanism. It is thus tempting to suggest that, as in animal models, lactic acid may be a damaging factor for colonic mucosa in patients with UC.
References 1. Roediger WEW. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. Gut 1980;21:793-8. 2. Roediger WEW. The colonic epithelium in ulcerative colitis: an energy deficiency disease. Lancet 1980;ii:712-5. 3. Roediger WEW, Heyworth M, Willoughby P, Piris 1. Moore A, Truelove SC. Luminal ions and short chain fatty acids as markers of functional activity of the mucosa in ulcerative colitis. J Clin Pathol 1982;35:323-6. 4. Vernia P, Latella G, Magliocca FM, Barbetti F, Caprilli R. Fecal lactate in ulcerative colitis (abstr). Gastroenterology 1986;90:1680. 5. Roediger WEW, Lawsong M1. Kwok V, Kerr Grant A, Pannall PRo Colonic bicarbonate output as a test of disease activity in ulcerative colitis. J Clin PathoI1984;37:704-7. 6. Caprilli R, Frieri G, Latella G, Santoro ML, Vernia P. Faecal excretion of bicarbonate in ulcerative colitis. Digestion 1986; 35:136-42. 7. Saunders DR, Sillery J. Effect of lactate and pH on structure and function of rat intestine. Dig Dis Sci 1982;27:33-41. 8. Caprilli R. Sebastiani R. Palumbo G, et al. Colite sperimentale da acido lattico. Proceedings of the XXVI Congress of the Italian Society of Gastroenterology, Sorrento, November 5-7, 1987:54. 9. Schachter H, Kirsner SB. Definitions of inflammatory bowel disease of unknown etiology. Gastroenterology 1975;68:591600. 10. Truelove SC, Witts LJ. Cortisone in ulcerative colitis: final report on a therapeutic trial. Br Med J 1955;2:1041-8.
GASTROENTEROLOGY Vol. 95, No.6
11. Vernia P, Breuer RI, Gnaedinger A, Latella G, Santoro ML. The composition of fecal water. Comparison of "in vitro" dialysis with ultrafiltration. Gastroenterology 1984;86:155761. 12. Holdeman LV, Cato EP, Moore WECo Chromatographic procedures for analysis of acid and products. In: Anaerobe laboratory manual. 4th ed. Anaerobe Laboratory of the Virginia Polytechnic Institute and State University. Blacksburg, Va.: Southern Printing Co., 1977:134-7. 13. Gutman 1, Wahlefeld AW. Lactate determination with lactate dehydrogenase and NAD. In: Bergmeyer HU, ed. Methods of enzymatic analysis. New York, London: Academy, 1974: 1464-8. 14. Caprilli R, Sopranzi N, Colaneri 0, Levi della Vida MV, De Magistris L. Salt losing diarrhoea in idiopathic proctocolitis. Scand J GastroenteroI1978;13:331-5. 15. Schilli R, Breuer R1, Klein F, et al. Comparison of the composition of fecal fluid in Crohn's disease and ulcerative colitis. Gut 1982;23:326-32. 16. Ellestad-Sayed JJ, Nelson RA, Martin A, Palmer M, Soule E. Pantothenic acid, coenzyme A, and human chronic ulcerative and granulomatous colitis. Am J Clin Nutr 1976;29:1333-8. 17. Nelson RA. Intestinal transport, coenzyme A, and colitis in pantothenic acid deficiency. Am J Clin Nutr 1968;21:495501. 18. Montgomery RD, Frazer AC, Hood C, Goodhart JM, Holland MR, Schneider R. Studies of intestinal fermentation in ulcerative colitis Gut 1968;9:521-6. 19. Mc Neil Nl. The effect of luminal pH on large intestinal absorption in ulcerative proctocolitis. Dis Colon Rectum 1986;29:814-6. 20. Russel JB, Sharp WM, Baldwin RL. The effect of pH on maximum bacterial growth rate and its possible role as a determinant of bacterial competition in the rumen. J Anim Sci 1979;48:251-5. 21. Edwards CA, Duerden BI, Read NW. The effects of pH on colonic bacterial growth in continuous culture. J Med Microbioi 1985;19:169-80. 22. Miller TL, Wolin MJ. Fermentations by saccharolytic intestinal bacteria. Am J Clin Nutr 1979;32:164-172. 23. Perman JA, Modler S, Olson AC. Role of pH in production of hydrogen from carbohydrates by colonic bacterial flora. J Clin Invest 1981;67:643-50. 24. Vand der Wiel-Korstanje SAA, Winkler KC. The faecal flora in ulcerative colitis. J Med Microbiol 1975;8:491-501.
Received November 23, 1987. Accepted July 18, 1988. Address requests for reprints to: P. Vernia, M.D., Cattedra di Gastroenterologia (1), Universita "La Sapienza" Roma, 2a Clinic a Medica, Policlinico Umberto I, 00161 Rome, Italy.
Fecal Lactate and Ulcerative Colitis PIERO VERNIA, RENZO CAPRILLI, GIOVANNI LATELLA, FABRIZIO BARBETTI, FABIO M. MAGLIOCCA, and MAURO CITTADINI
Department of Gastroenterology, Universita "La Sapienza," Rome, and Department of Gastroenterology, Universita de L'Aquila, L'Aquila, Italy
Impaired metabolism of short-chain fatty acids, as well as a modified fecal ionogram, have been reported in ulcerative colitis. Fecal water samples from 62 patients with ulcerative colitis were analyzed in the present investigation to evaluate changes in SCFAs and lactic acid in relation to activity and severity of disease. Short-chain fatty acid levels were high in quiescent and mild disease (162.6 ± 63.6 and 147.8 ± 63.2 mMlL, respectively), but significantly decreased in the severe form (64.7 ± 46.9 mM/L). Lactate showed a progressive increase from mild colitis (3.0 ± 1.8 mM/L) to severe colitis (21.4 ± 18.6 mM/L). It thus appears that mild colitis displayed a fecal pattern characterized by normal pH and bicarbonate, slightly impaired electrolyte handling, high short-chain fatty acid values, and only moderately increased lactate. Severe colitis, on the other hand, was characterized by low fecal pH, bicarbonate, and potassium, high sodium and chloride, low short-chain fatty acid levels, and very high lactate levels. A critical lowering of intraluminal pH, which shifts bacterial metabolism from shortchain fatty acid to lactate production, may be responsible for the intraluminal pooling of lactate.
S
hort-chain fatty acids (SCFAs), products of bacterial fermentation of unabsorbed carbohydrates, represent a primary energy source for colonic mucosal cells (1). Impaired absorption or utilization of SCFAs, or both, by the colonic mucosa has recently been described in patients with ulcerative colitis (UC) (2,3), suggesting a change in colonocyte metabolism. In preliminary studies we reported an abnormal fecal organic ionogram in UC, mainly characterized by an increase in lactate (4). The fecal excretion of bicarbonate is reduced and fecal pH is low in patients with active UC (5,6). Interestingly, in experimental studies on rats, the association of high intrduminal content of lactate (100 mM/L) and low
pH «4) reduced water and electrolyte absorption and induced colonic mucosal damage (7). Furthermore, in our laboratory mucosal alterations strictly resembling those of human UC were induced in rats by the infusion of lactic acid at a concentration of 25 mM/L, which is of the same order as those found in the stools of patients with UC (8). It seems, therefore, that the change in organic anion metabolism, associated with low intraluminal pH, could be involved in the onset or deterioration of colitic lesions. The aim of the present study was, therefore, to evaluate the changes in fecal pH, SCFAs, and lactate in relation to the activity and severity of colitis.
Patients and Methods Patients The study was carried out on 62 patients with UC (5 quiescent, 57 active disease) admitted to the GI Unit of the University of Rome during the period 1982-86, and 29 healthy controls. Diagnosis was based on clinical, radiologic, endoscopic, and histologic findings (9). The severity of the disease was assessed according to the criteria of Truelove and Witts (10). Colitis was classified as severe in 20, moderate in 23, and mild in"14 patients. The extent of the lesions was established from radiologic (doublecontrast barium enema), endoscopic (total colonoscopy), and histologic findings. Twenty-four patients whose lesions spread beyond the transverse colon were considered to have total colitis. The lesions did not reach the midtransverse colon in the 19 cases classified as left-sided colitis. Thirteen patients had proctosigmoiditis. Data from patients with quiescent colitis were evaluated separately. No patient was taking oral corticosteroids or using local corticosteroids before the stool collection.
Abbreviutions used in this puper: SCF A, short-chain fatty acid; UC, ulcerative colitis. © 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
FECAL ORGANIC ANIONS IN DC
December 1988
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Table 1. Fecal Wet Weight, pH, and Electrolytes in Relation to the Severity of Colitis Colitis
Fecal weight pH Na+ K+ ClCa 2+ Mg2+ K+/Na+
Controls
Quiescent
Mild
Moderate
Severe
(29)
(5)
(14)
(23)
(20)
149.2 6.8 18.2 61.2 13.8 26.0 41.6 6.9
± ± ± ± ± ± ± ±
101.3 0.7 17.1 25.1 10.8 18.8 33.2 6.1
234.9 7.2 32.5 52.5 28.3 27.6 28.0 2.7
± ± ± ± ± ± ± ±
221.1 6.4 30.4 51.6 23.1 44.4 53.3 3.1
123 0.7 14.9 18.8 18.3 26.0 22.4 3.1
± ± ± ± ± ± ± ±
82.7 d
346.3 6.3 47.6 39.8 36.3 30.8 25.6 1.1
0.8 20.2 d 22.0 14.8 d 29.6 42.8 3.1c
± ± ± ± ± ± ± ±
165.4" 0.5 b 23.5" 13.7 b 17.9" 22.0 7.6 c 0.8"
± ± ± ± ± ± ± ±
541.0 6.2 62.7 35.7 50.4 16.8 14.0 0.6
418.9" O.4 b 22.8" 17.9" 23.9" 10.4 d 12.4 b 0.5"
Data expressed in mM/L; fecal weight in grams per 24 h. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.0001, b P < 0.001, C P < 0.01, d P < 0.05 versus controls.
Fecal Analysis Twenty-four-hour stool specimens were collected in preweighed, air-tight plastic cans upon admission. Thiomersal (15 ml, 1: 10,000 solution) was added immediately after the first evacuation to minimize bacterial overgrowth. Fecal water was extracted using an in vitro dialysis method and analyzed immediately or frozen and stored at a temperature of -20°C (11). Organic anions were analyzed by gas-liquid chromatography (12), and their concentration was calculated against that of standard solutions of the following acids: acetic, propionic, isobutyrie, butyric, isovaleric, valerie, isocaproie, and caproic. Concentrations of L- and D-lactic acid were measured by an enzymatic method using specific L- and D-lactate dehydrogenase (Boehringer Biochem) (13). Sodium, potassium, calcium, and magnesium concentrations were measured by flame photometry and chloride by potentiometric titration. The stool pH was measured on freshly passed stools by a glass electrode connected to a Beckman pH-meter (Beckman Instruments, Fullerton, Calif.).
Statistical Analysis Data were analyzed by Student's t-test for unpaired data. Correlation analysis was performed in normal controls and in patients with UC, considering all patients as a single population, and in each individual subgroup. Despite the fact that only correlations showing a p value
< 0.001 were taken into account, many variables were found to be significantly correlated. A multivariate analysis (Forward Multiple Linear Regression Test) was thus performed, considering as dependent variables the 24-h fecal wet weight, fecal pH, and concentration and output of sodium, potassium, chloride, lactic acid, and SCFAs. Only those parameters significantly influencing the dependent variable (p < 0.001) were taken into consideration.
Results The fecal wet weight and pH and inorganic component of fecal water are shown in Tables 1 and 2. Stool weight and sodium and chloride concentrations increased with the severity and extent of the disease, whereas the potassium level, potassium to sodium ratio, and pH showed an opposite trend. Similar modifications were also found in patients with quiescent disease. Concentrations of the organic anions of fecal water are shown in Tables 3 and 4. All SCF As were found to be slightly increased in quiescent and mild colitis, almost normal in moderate disease, but significantly reduced in severe colitis. Lactic acid concentration, on the other hand, showed an extremely significant increase in all patients with colitis. The increase of lactic acid was mainly due to the L-form of the acid (Figure 1). This
Table 2. Fecal Wet Weight, pH, and Electrolytes in Relation to the Extent of the Lesions
Fecal weight pH Na K ClCa 2+ Mg2+ K+/Na+
Controls
Proctosigmoiditis
Left-sided colitis
Total colitis
(29)
(13)
(20)
(24)
149.2 6.8 18.2 61.2 13.8 26.0 43.6 6.9
± ± ± ± ± ± ± ±
101.3 0.7 17.1 25.1 10.8 18.8 33.2 6.1
234.9 6.1 32.5 52.5 28.3 28.8 32.4 2.7
± ± ± ± ± ± ± ±
123.2" O.4 b 14.9 c 18.4 18.3 c 32.4 23.6 3.1 b
327.6 5.9 46.4 37.2 38.5 24.4 46.4 1.1
± ± ± ± ± ± ± ±
132.4° 0.5 b 23.1" 15.9" 21.8 0 12.4 48.8 0.7 0
510.7 6.5 59.4 38.5 42.9 33.6 31.2 1.1
± ± ± ± ± ± ± ±
398" 0.4 26.8" 18.10 23.2" 26.4 26.8 1.3"
Data expressed in mM/L; fecal weight in grams per 24 h. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.0001, b P < 0.001, C P < 0.01 versus controls.
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GASTROENTEROLOGY Vol. 95, No.6
Table 3. Organic Anions in Relation to the Severity of the Colitis Colitis Controls (29) Acids Acetic Propionic Isobutyric Butyric Isovaleric Valeric Isocaproic Caproic Total SCFAs L-Lactic D-Lactic Total lactic
71.5 17.3 1.9 13.7 2.4 2.3 0.8 0.3 111.4
± ± ± ± ± ± ± ± ±
Quiescent (5)
47.3 8.4 1.0 8.4 1.8 1.2 0.2 0.4 58.6
0.2 ± 0.1 0.3 ± 0.2 0.5 ± 0.2
113.4 23.3 2.4 16.3 3.1 2.7 0.03 0.2 162.6
± ± ± ± ± ± ± ± ±
Mild (14)
60.7 10.5 1.1 4.0 1.8 2.0 0.04 0.1 63.6
96.3 28.2 2.1 17.7 2.5 2.5 0.1 0.3 147.8
± ± ± ± ± ± ± ± ±
Moderate (23)
36.6 14.8 d 1.9 11.1 2.6 3.9 0.2 0.5 63.2
63.9 22.9 1.3 14.6 1.4 1.7 0.7 0.1 106.0
2.0 ± 1.6 b 1.0 ± 1.2 3.0 ± 1.80
0.6 ± 0.8 0.9 ± 1.5 1.5 ± 2.4
± ± ± ± ± ± ± ± ±
35.7 30.0 0.8 d 13.2 0.9 d 1.5 0.2 0.2 74.1
7.8 ± 7.20 1.1 ± 1.5 8.9 ± 7.0 0
Severe (20) 47.8 10.2 0.9 4.3 1.2 0.6 0.3 0.07 64.7
± ± ± ± ± ± ± ± ±
32.1 d 8.7 b 1.3 b 5.0 0 1.7 d 0.7 0 1.0 O.ld 46.9 b
20.1 ± 13.3 0 1.3 ± 11.3 C 21.4 ± 18.60
SCFAs, short-chain fatty acids. Data expressed in mMIL. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.001, b P < 0.005, C P < 0.01, d P < 0.02 versus controls.
finding was also present in patients with quiescent colitis, although values were not statistically significant.
Correlation Analysis in Normal Subjects Linear regression analysis. Fecal weight showed a positive linear correlation with sodium (r = 0.62) and chloride (r = 0.54), but a negative correlation with fecal pH (r = -0.47). Multiple linear regression analysis. Fecal wet weight was significantly influenced only by sodium concentration and fecal pH. Sodium output was related to that of butyric acid, potassium, chloride, and lactic acid.
Correlation Analysis in Patients With Ulcerative Colitis Linear regression analysis. The 24-h fecal wet weight showed good correlation values with the concentration of sodium (r = 0.48) and the output of sodium (r = 0.95), chloride (r = 0.90), potassium (r = 0.69), lactic acid (r = 0.58), and SCFAs (r = 0.51). The concentrations of SCFAs showed negative correlations with sodium (r = -0.54) and chloride concentrations (r = -0.51), but a positive correlation with that of potassium (r = 0.42). Positive correlations were also found between lactic acid output and fecal weight (r = 0.58), and the output of sodium (r = 0.58), chloride (r = 0.49), and acetic acid (r = 0.42). Multiple linear regression. Once the independent variable "sodium" was taken into account, no
Table 4. Organic Anions in Relation to the Extent of the Lesions Controls (29) Acids Acetic Propionic Isobutyric Butyric Isovaleric Valeric Isocaproic Caproic Total SCFAs L-Lactic D-Lactic Total lactic
71.5 17.3 1.9 13.7 2.4 2.3 0.8 0.3 111.4
± ± ± ± ± ± ± ± ±
47.3 8.4 1.0 8.4 1.8 1.2 0.2 0.4 58.6
0.2 ± 0.1 0.3 ± 0.2 0.5 ± 0.2
Proctosigmoiditis (13) 81.3 31.0 1.6 15.3 2.1 2.4 0.2 0.3 129.8
± ± ± ± ± ± ± ± ±
44.4 39.0 1.6 14.9 2.3 3.1 0.5 0.6 98.4
8.1 ± 12.3 c 1.4 ± 1.1 9.5 ± 10.9 b
Left-sided colitis (20) 67.8 16.5 1.3 11.9 1.6 1.3 0.3 0.08 101.4
± ± ± ± ± ± ± ± ±
37.3 13.0 1.1 d 9.7 1.6 l.4 b 1.1 O.ld 56.1
9.6 ± 10.8 b 1.1 ± 1.4 c 10.7 ± 10.3 0
Total colitis (24) 56.8 16.1 1.3 9.6 1.5 1.2 0.4 0.1 86.8
± ± ± ± ± ± ± ± ±
35.9 12.0 1.5 11.2 1.7 d 2.2 d 0.2 0.2 58.3
12.9 ± 12.9 b 1.2 ± 1.7C 14.1 ± 17.80
SCFAs, short-chain fatty acids. Data expressed in mMIL. Mean values ± SD. Numbers in parentheses represent the number of patients in each group. Student's t-test: a p < 0.001, b P < 0.01, C P < 0.02, d P < 0.05 versus controls.
FECAL ORGANIC ANIONS IN UC
December 1988
•
' - -_ _....II D-LACTIC ACID
25
wn;:m
L-LACTIC ACID
• p<
0.0001
20
15
10
5
o
.
:::::::::
T :::::::::
r----L---;:::::::::: ......... ................. ........ ........ ........ .. ........ ................. ........ ........ . .................. :::::::: :::::::::: T :::::::: :::::::::: .....--'---1;.. .. .. •• .. . . . . . . . . .................. ............... .. r--_TL--t.:;~~ ••••••••••••••••• •••••• , ••• ........................... ......... ........ -:::::::::
.
CONTROLS QUIESCENT COLITIS
MILO COLITIS
,,"",-rn-rnM/•••••••••
MODERATE COLITIS
SEVERE COLITIS
Figure 1. Lactic acid concentration in the stools of patients with ulcerative colitis and normal controls. Mean values (mM/L) ± SEM (vertical bars). Probability values express differences versus controls.
other variable significantly influenced stool weight. The 24-h losses of sodium, on the other hand, were related with decreasing statistical weight to chloride output, stool weight, and potassium and lactic acid output. Chloride losses were shown to be influenced mainly by the output of sodium, but also by the output of lactic acid and butyric acid. Besides all individual SCFAs, only fecal pH was shown to influence SCFA concentration, whereas the concentration of lactic acid varied in relation to those of sodium and butyric acid. Output of lactic acid was again influenced by that of sodium and butyric acid, but also by that of chloride and acetic acid.
Discussion As already reported in previous investigations (14,15), the stools of patients with active UC are characterized by high 24-h weight, high concentrations of sodium and chloride, low concentrations of potassium, reduced potassium to sodium ratio, and low pH. These changes progressively increase with the severity of the disease and extent of the lesions. Similar changes are also present in patients with
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quiescent colitis, with the exception of pH, which is normal. As far as the organic component is concerned, fecal SCFAs are high in patients with mild disease, do not differ from controls in moderate disease, and are significantly low in severe cases. Lactate, which is present in negligible concentrations in the fecal water of normal subjects and patients with quiescent colitis, was found to be significantly increased in patients with active disease, reaching very high concentrations in severe colitis. The high concentration of SCFAs observed in mild colitis may be attributed to their impaired utilization by the colonic mucosa as suggested by Roediger (2,3). The decreased amount of coenzyme A in the mucosa of patients with UC reported some years ago (16) may account for the reduced oxidation of SCFAs (3). This hypothesis is also supported by the close similarity existing between colonic lesions in UC and those reported in experimental pantothenic acid deficiency colitis in pigs (17). It is well known that coenzyme A intervenes at different levels of intracellular metabolic pathways, i.e., l3-oxidation of SCFAs and the Krebs' cycle. Its deficit, therefore, results in an impaired utilization of SCFAs, which are the main energetic fuel of the colonic mucosa, with consequent impairment of all intracellular energydependent processes such as electrolyte exchanges and mucus and structural protein synthesis. Other factors, such as increased production of SCFAs due to less effective absorption of dietary carbohydrates (18) or diminished SCFA absorption by the inflamed mucosa (19), cannot be ruled out. In our series of patients with severe colitis, however, the fecal concentration of SCF As was significantly lower than in normal controls, suggesting that factors other than impaired SCF A absorption and utilization are present. It is noteworthy that the reduction of SCFAs was paralleled by increasing lactate concentrations and lowering of pH. The low fecal pH, due to reduced colonic bicarbonate secretion (5,6), likely reflects the low intraluminal pH. An acidic intraluminal environment is unfavorable for some SCFA-producing strains but not for lactateproducing strains, and shifts bacterial metabolic pathways from SCFA to lactate production (20-24). Other mechanisms may also lead to increased lactate production in colitis. The increased intraluminal oxygen concentration, resulting from profuse bleeding, favors facultative anaerobic strains such as lactobacilli and streptococci (24), which are lactic acid producers. Finally, the increased mucus production, the disruption of the colonic mucosal cell lining, and the exposure of mesenchymal polysaccharides (hyaluronic acid) to intraluminal bacteria may also lead to lactate production (24).
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VERNIA ET AL.
On the basis of stool analysis, two different phases may be recognized in the clinical course of uc. Mild colitis displays a fecal pattern characterized by normal fecal pH, normal excretion of bicarbonate, high concentrations of SCFAs, and only mildly increased lactate. Severe colitis is characterized by low fecal pH, reduced excretion of bicarbonate, low concentrations of SCFAs, and markedly increased lactic acid. The critical lowering of bicarbonate secretion and intracolonic pH may be responsible for the altered fecal water composition found in severe colitis. Low pH in fact reduces the availability of SCFAs, and at the same time enhances the production of lactic acid. This in turn may contribute to colonic damage through a direct mechanism. It is thus tempting to suggest that, as in animal models, lactic acid may be a damaging factor for colonic mucosa in patients with UC.
References 1. Roediger WEW. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. Gut 1980;21:793-8. 2. Roediger WEW. The colonic epithelium in ulcerative colitis: an energy deficiency disease. Lancet 1980;ii:712-5. 3. Roediger WEW, Heyworth M, Willoughby P, Piris 1. Moore A, Truelove SC. Luminal ions and short chain fatty acids as markers of functional activity of the mucosa in ulcerative colitis. J Clin Pathol 1982;35:323-6. 4. Vernia P, Latella G, Magliocca FM, Barbetti F, Caprilli R. Fecal lactate in ulcerative colitis (abstr). Gastroenterology 1986;90:1680. 5. Roediger WEW, Lawsong M1. Kwok V, Kerr Grant A, Pannall PRo Colonic bicarbonate output as a test of disease activity in ulcerative colitis. J Clin PathoI1984;37:704-7. 6. Caprilli R, Frieri G, Latella G, Santoro ML, Vernia P. Faecal excretion of bicarbonate in ulcerative colitis. Digestion 1986; 35:136-42. 7. Saunders DR, Sillery J. Effect of lactate and pH on structure and function of rat intestine. Dig Dis Sci 1982;27:33-41. 8. Caprilli R. Sebastiani R. Palumbo G, et al. Colite sperimentale da acido lattico. Proceedings of the XXVI Congress of the Italian Society of Gastroenterology, Sorrento, November 5-7, 1987:54. 9. Schachter H, Kirsner SB. Definitions of inflammatory bowel disease of unknown etiology. Gastroenterology 1975;68:591600. 10. Truelove SC, Witts LJ. Cortisone in ulcerative colitis: final report on a therapeutic trial. Br Med J 1955;2:1041-8.
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11. Vernia P, Breuer RI, Gnaedinger A, Latella G, Santoro ML. The composition of fecal water. Comparison of "in vitro" dialysis with ultrafiltration. Gastroenterology 1984;86:155761. 12. Holdeman LV, Cato EP, Moore WECo Chromatographic procedures for analysis of acid and products. In: Anaerobe laboratory manual. 4th ed. Anaerobe Laboratory of the Virginia Polytechnic Institute and State University. Blacksburg, Va.: Southern Printing Co., 1977:134-7. 13. Gutman 1, Wahlefeld AW. Lactate determination with lactate dehydrogenase and NAD. In: Bergmeyer HU, ed. Methods of enzymatic analysis. New York, London: Academy, 1974: 1464-8. 14. Caprilli R, Sopranzi N, Colaneri 0, Levi della Vida MV, De Magistris L. Salt losing diarrhoea in idiopathic proctocolitis. Scand J GastroenteroI1978;13:331-5. 15. Schilli R, Breuer R1, Klein F, et al. Comparison of the composition of fecal fluid in Crohn's disease and ulcerative colitis. Gut 1982;23:326-32. 16. Ellestad-Sayed JJ, Nelson RA, Martin A, Palmer M, Soule E. Pantothenic acid, coenzyme A, and human chronic ulcerative and granulomatous colitis. Am J Clin Nutr 1976;29:1333-8. 17. Nelson RA. Intestinal transport, coenzyme A, and colitis in pantothenic acid deficiency. Am J Clin Nutr 1968;21:495501. 18. Montgomery RD, Frazer AC, Hood C, Goodhart JM, Holland MR, Schneider R. Studies of intestinal fermentation in ulcerative colitis Gut 1968;9:521-6. 19. Mc Neil Nl. The effect of luminal pH on large intestinal absorption in ulcerative proctocolitis. Dis Colon Rectum 1986;29:814-6. 20. Russel JB, Sharp WM, Baldwin RL. The effect of pH on maximum bacterial growth rate and its possible role as a determinant of bacterial competition in the rumen. J Anim Sci 1979;48:251-5. 21. Edwards CA, Duerden BI, Read NW. The effects of pH on colonic bacterial growth in continuous culture. J Med Microbioi 1985;19:169-80. 22. Miller TL, Wolin MJ. Fermentations by saccharolytic intestinal bacteria. Am J Clin Nutr 1979;32:164-172. 23. Perman JA, Modler S, Olson AC. Role of pH in production of hydrogen from carbohydrates by colonic bacterial flora. J Clin Invest 1981;67:643-50. 24. Vand der Wiel-Korstanje SAA, Winkler KC. The faecal flora in ulcerative colitis. J Med Microbiol 1975;8:491-501.
Received November 23, 1987. Accepted July 18, 1988. Address requests for reprints to: P. Vernia, M.D., Cattedra di Gastroenterologia (1), Universita "La Sapienza" Roma, 2a Clinic a Medica, Policlinico Umberto I, 00161 Rome, Italy.