LETTERS TO THE EDITOR STARCH DIGESTIBILITY IN VIVO AND IN VITRO
To The Editor: Using the breath hydrogen technique, we have shown that a freeze-thaw cycle raises small intestinal malabsorption and subsequent colonic fermentation of potato starch; this effect was partially reversible by reheating (1). In an extension of the in vivo study, we have now performed in vitro experiments on starch digestibility. Following the protocol of the study above, samples of freshly boiled potatoes (A), boiled/frozen/thawed potatoes (B), and boiled/frozen/thawed/reheated potatoes (C) were homogenized and suspended in acetate buffer (100 mrnol/liter). Pancreatin (amylase activity 1000 units/ml) and amyloglucosidase (1000 units/ml) were added, and the samples incubated at 37~ C for 180 rain with mixing every 5 rain. Subsamples were removed at 30, 60, 120, and 180 min; deproteinized; and glucose measured with a glucose oxidase assay. A factor of 0.9 was used to convert the glucose measured to starch digested. Total starch was determined after dispersion with 7 M KOH and enzymatic digestion at 60~ C. Results are shown in Table 1. It is concluded that a freeze-thaw cycle reduces both the speed of starch digestion and overall digestibility. Similar to the in vivo data, this effect is partly reversible by reheating. This observation may be due to the formation of retrograded amylose (RS 3 according to Englyst) (2) and probably other poorly digestible products. The availability of carbohydrates for fermentation in the large bowel is thought to be a key regulating factor of the colonic environment (3). STEFAN CHRISTL, MD WOLFGANG SCHEPPACH, M D
Department of Medicine University of Wiirzburg Josef-Schneider-Strasse 2 D-8700 Wiirzburg, Germany
REFERENCES 1. Scheppach W, Bach M, Bartram P, Christi S, Bergthaller W, Kasper H: Colonic fermentation of potato starch after a freeze-thaw cycle. Dig Dis Sci 36:1601-1605, 1991 2. Englyst HN, Kingman SM: Dietary fiber and resistent starch. A nutritional classification of plant polysaccharides. In Dietary Fiber: Chemistry, Physiology and Health Effects. D Kritchevsky, JA Anderson (eds). New York, Plenum Publishing Corp, 1990, pp 49-65
156
TABLE 1. STARCHDIGESTED (g) MEASUREDAS GLUCOSE RELEASED * 0.9 AFTER 30, 60, 120, 180 MINUTES OF INCUBATION, AND TOTAL STARCHAFTER DISPERSION W I T H KOH* Incubation time (min) 30
A B C
44.5bc (---1.7) 27.3ac (-+ 1.8) 38.2ab (-+2.2)
60
64. lbc (-+2.1) 42.3ac (-+ 1.7) 58.4ab (-+2.6)
120
66.4bc (-+2.5) 52.8ac (-+2.0) 61.2ab (-+2.7)
180
67.3b (-+2.3) 60.9ac (-+ 1.9) 65.0b (-+2.7)
Total starch
69.4 (---1.3) 69.2 (-- 1.2) 69.4 (-+2.4)
*Mean of eight experiments --_ sD; a--c indicate significant differences (P < 0.05, t test) towards groups with the same letter.
3. Cummings JH, Englyst HN: Measurement of starch fermentation in the human large intestine. Can J Physiol Pharmacol 69:121-129, 1991
PREVALENCE OF ANTI-HEPATITIS C VIRUS ANTIBODIES AMONG PATIENTS WITH ALCOHOLIC LIVER DISEASE, SUPPLEMENTED BY 4-RIBA
To The Editor: Early reports have shown a high prevalence of antibodies to the hepatitis C virus among patients with alcoholic liver disease (1-3). This prevalence seems to be correlated with the severity of liver injury. The presence of anti-HCV antibodies was detected by using an e n z y m e immunoassay (ELISA) with a nonstructural protein (c100-3) as antigen. False positive results of this test have been reported in various chronic liver diseases (4). In autoimmune chronic active hepatitis, the optical density values in the ELISA test correlated closely with serum globulins and IgG concentrations (5). Hyperglobulinemia related to cirrhosis should be taken into account in the interpretation of ELISA in patients with alcoholic cirrhosis. One remedy to detect the "true positive" patients was to use the new recombinant immunoblot assay detecting four types of antibodies one to structural protein (c22c) and three to nonstructural protein (5-I-1, c100-3, c33c). We prospectively investigated 113 consecutive patients (mean age 52.8 years) with alcoholic liver disease (80 men and 33 women). Eighty-six patients had liver cirrhosis and 27 had only alcoholic hepatitis. Daily alcohol consumption for all patients was higher than 80 g ethanol for at least five years. Patients with hepatocellular carcinoma were excluded from this study. The "Ortho-HCV" enDigestive Diseases and Sciences, Vol. 37, No. 1 (January 1992)
LETTERS TO T H E EDITOR
zyme-linked immunosorbent assay (ELISA, Ortho Diagnostics) was used to detect anti-HCV antibodies. Patients with anti-HCV antibodies detected by ELISA were tested by using a four-antigen recombinant immunoblot assay (4-RIBA, Ortho Diagnostics). Patients' sera were also tested for HBs antigen (HBsAg), antibody to hepatitis B core antigen (antiHBc), and antibody to hepatitis B surface antigen (anti-HB~), by radioimmunoassay (Abbott Laboratories). Anti-HCV antibodies were detected by ELISA in 7.9% of patients; 3.5% of patients with alcoholic hepatitis and 10.5% of those with liver cirrhosis. There was no significant difference between these two groups. Anti-HBV markers were found in 30% of all patients without any significant difference between patients with hepatitis or cirrhosis. No relationship could be demonstrated between the positivity of anti-HBV markers and the presence or absence of anti-HCV antibodies, respectively (20% and 31%). Six of 10 sera reactive in ELISA were positive by 4-RIBA. Two patients were negative for 4-RIBA test, two were indeterminate. The incidence of blood transfusion was 33% (2/6) of patients with positive anti-HCV antibodies detected either by ELISA and RIBA, and 26% (28/107) of those without anti-HCV antibodies. This difference was not significant. In this study, the prevalence of anti HCV anti9bodies (7.9%) was lower than in most previous studies. In fact, the results previously reported were distributed over a large range (5-38%) (1-3) and were determined in retrospective studies. Although in our population the prevalence of antiHCV antibodies was low, it was seven times greater than the prevalence observed in blood donors in France. In order to estimate the prevalence of prior or current hepatitis C virus infection in alcoholic patients, we tried to demonstrate the presence of antibodies to the products of one or more hepatitis C genes other than c100-3. Thus in our series, the true prevalence of anti-HCV antibodies should be corrected and estimated to be around 5.3%. The two negative patients had an immunoglobulin concentration higher than 40 g/100 ml; the six concordant patients for the two tests had a rate lower than 30 g/100 ml. For the two remaining patients, with an indeterminate 4-RIBA test, serum immunoglobulin levels were 25 and 15 g/100 ml respectively. We
Digestive Diseases and Sciences, Vol. 37, No. 1 (January 1992>
think that the difference observed in the prevalence of anti-HCV antibodies in our series compared to others could be explained, on the one hand, by false-positive ELISA results due to a nonspecific IgG component adhering to the ELISA plates and, on the other, by testing fresh serum samples in this study. Since indeed, it has been suggested that frozen stored serum may give false-positive reactions in an enzyme immunoassay for c100-3 antibody (6). In conclusion, the rate of false-positive first-generation ELISA tests could be estimated in the range of 20-40% in patients with alcoholic liver diseases. As previously observed, this result is closely correlated to serum immunoglobulin concentration of these patients. The HCV-RNA detection after amplification in sera or in hepatic tissues should be helpful in some indeterminate cases. P. LAURENT-PUIG, MD E. DUSSAIX, MD Y. LECOZ, MD P. MA~TES, MD C. BUFFET, MD Service des maladies du foie et de l'appareil digestif Laboratoire de virologie H6pital de Bic~tre 78 Avenue du Gdndral Leclerc 94270 Le Kremlin Bic~tre, France REFERENCES 1. Pares A, Barrera JM, Caballeria J, et al: Hepatitis C virus antibodies in chronic alcoholic patients: Association with severity of liver injury. Hepatology 12:1295-1299, 1990 2. Jacyna MR, Goldin R, McSween R, Thomas HC: Significance of anti-HCV antibodies with alcoholic liver disease from England and Scotland. Gastroenterology 100:A756, 1990 3. Btuix J, Barrera JM, Cavet X, Ercilla G, Costa J, SanchezTapias JM, Ventura M, Vail M, Bruguera M, Bru C, Castillo R, Rodes J: Prevalence of antibodies to hepatitis C virus in a Spanish patient with hepatocellular carcinoma and hepatitis cirrhosis. Lancet 2:1004-1008, 1989 4. Gray JJ, Wreghitt TG, Friend PJ, Wight DGD, Sundaresan V, Calne RY: Differentiation between specific and nonspecific hepatitis C antibodies in chronic liver disease. Lancet 335:610-619, 1990 5. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R: Hepatitis C virus antibodies in chronic active hepatitis: Pathogenic factor or false-positive result. Lancet 335:754-757, 1990 6. Mortimer PP, Cohen BJ, Litton PA, Vandervelde EM, Bassendine MF, Brind AM, Hambling MH: Hepatitis C virus antibody. Lancet 2:798, 1989
157
To The Editor: Using the breath hydrogen technique, we have shown that a freeze-thaw cycle raises small intestinal malabsorption and subsequent colonic fermentation of potato starch; this effect was partially reversible by reheating (1). In an extension of the in vivo study, we have now performed in vitro experiments on starch digestibility. Following the protocol of the study above, samples of freshly boiled potatoes (A), boiled/frozen/thawed potatoes (B), and boiled/frozen/thawed/reheated potatoes (C) were homogenized and suspended in acetate buffer (100 mrnol/liter). Pancreatin (amylase activity 1000 units/ml) and amyloglucosidase (1000 units/ml) were added, and the samples incubated at 37~ C for 180 rain with mixing every 5 rain. Subsamples were removed at 30, 60, 120, and 180 min; deproteinized; and glucose measured with a glucose oxidase assay. A factor of 0.9 was used to convert the glucose measured to starch digested. Total starch was determined after dispersion with 7 M KOH and enzymatic digestion at 60~ C. Results are shown in Table 1. It is concluded that a freeze-thaw cycle reduces both the speed of starch digestion and overall digestibility. Similar to the in vivo data, this effect is partly reversible by reheating. This observation may be due to the formation of retrograded amylose (RS 3 according to Englyst) (2) and probably other poorly digestible products. The availability of carbohydrates for fermentation in the large bowel is thought to be a key regulating factor of the colonic environment (3). STEFAN CHRISTL, MD WOLFGANG SCHEPPACH, M D
Department of Medicine University of Wiirzburg Josef-Schneider-Strasse 2 D-8700 Wiirzburg, Germany
REFERENCES 1. Scheppach W, Bach M, Bartram P, Christi S, Bergthaller W, Kasper H: Colonic fermentation of potato starch after a freeze-thaw cycle. Dig Dis Sci 36:1601-1605, 1991 2. Englyst HN, Kingman SM: Dietary fiber and resistent starch. A nutritional classification of plant polysaccharides. In Dietary Fiber: Chemistry, Physiology and Health Effects. D Kritchevsky, JA Anderson (eds). New York, Plenum Publishing Corp, 1990, pp 49-65
156
TABLE 1. STARCHDIGESTED (g) MEASUREDAS GLUCOSE RELEASED * 0.9 AFTER 30, 60, 120, 180 MINUTES OF INCUBATION, AND TOTAL STARCHAFTER DISPERSION W I T H KOH* Incubation time (min) 30
A B C
44.5bc (---1.7) 27.3ac (-+ 1.8) 38.2ab (-+2.2)
60
64. lbc (-+2.1) 42.3ac (-+ 1.7) 58.4ab (-+2.6)
120
66.4bc (-+2.5) 52.8ac (-+2.0) 61.2ab (-+2.7)
180
67.3b (-+2.3) 60.9ac (-+ 1.9) 65.0b (-+2.7)
Total starch
69.4 (---1.3) 69.2 (-- 1.2) 69.4 (-+2.4)
*Mean of eight experiments --_ sD; a--c indicate significant differences (P < 0.05, t test) towards groups with the same letter.
3. Cummings JH, Englyst HN: Measurement of starch fermentation in the human large intestine. Can J Physiol Pharmacol 69:121-129, 1991
PREVALENCE OF ANTI-HEPATITIS C VIRUS ANTIBODIES AMONG PATIENTS WITH ALCOHOLIC LIVER DISEASE, SUPPLEMENTED BY 4-RIBA
To The Editor: Early reports have shown a high prevalence of antibodies to the hepatitis C virus among patients with alcoholic liver disease (1-3). This prevalence seems to be correlated with the severity of liver injury. The presence of anti-HCV antibodies was detected by using an e n z y m e immunoassay (ELISA) with a nonstructural protein (c100-3) as antigen. False positive results of this test have been reported in various chronic liver diseases (4). In autoimmune chronic active hepatitis, the optical density values in the ELISA test correlated closely with serum globulins and IgG concentrations (5). Hyperglobulinemia related to cirrhosis should be taken into account in the interpretation of ELISA in patients with alcoholic cirrhosis. One remedy to detect the "true positive" patients was to use the new recombinant immunoblot assay detecting four types of antibodies one to structural protein (c22c) and three to nonstructural protein (5-I-1, c100-3, c33c). We prospectively investigated 113 consecutive patients (mean age 52.8 years) with alcoholic liver disease (80 men and 33 women). Eighty-six patients had liver cirrhosis and 27 had only alcoholic hepatitis. Daily alcohol consumption for all patients was higher than 80 g ethanol for at least five years. Patients with hepatocellular carcinoma were excluded from this study. The "Ortho-HCV" enDigestive Diseases and Sciences, Vol. 37, No. 1 (January 1992)
LETTERS TO T H E EDITOR
zyme-linked immunosorbent assay (ELISA, Ortho Diagnostics) was used to detect anti-HCV antibodies. Patients with anti-HCV antibodies detected by ELISA were tested by using a four-antigen recombinant immunoblot assay (4-RIBA, Ortho Diagnostics). Patients' sera were also tested for HBs antigen (HBsAg), antibody to hepatitis B core antigen (antiHBc), and antibody to hepatitis B surface antigen (anti-HB~), by radioimmunoassay (Abbott Laboratories). Anti-HCV antibodies were detected by ELISA in 7.9% of patients; 3.5% of patients with alcoholic hepatitis and 10.5% of those with liver cirrhosis. There was no significant difference between these two groups. Anti-HBV markers were found in 30% of all patients without any significant difference between patients with hepatitis or cirrhosis. No relationship could be demonstrated between the positivity of anti-HBV markers and the presence or absence of anti-HCV antibodies, respectively (20% and 31%). Six of 10 sera reactive in ELISA were positive by 4-RIBA. Two patients were negative for 4-RIBA test, two were indeterminate. The incidence of blood transfusion was 33% (2/6) of patients with positive anti-HCV antibodies detected either by ELISA and RIBA, and 26% (28/107) of those without anti-HCV antibodies. This difference was not significant. In this study, the prevalence of anti HCV anti9bodies (7.9%) was lower than in most previous studies. In fact, the results previously reported were distributed over a large range (5-38%) (1-3) and were determined in retrospective studies. Although in our population the prevalence of antiHCV antibodies was low, it was seven times greater than the prevalence observed in blood donors in France. In order to estimate the prevalence of prior or current hepatitis C virus infection in alcoholic patients, we tried to demonstrate the presence of antibodies to the products of one or more hepatitis C genes other than c100-3. Thus in our series, the true prevalence of anti-HCV antibodies should be corrected and estimated to be around 5.3%. The two negative patients had an immunoglobulin concentration higher than 40 g/100 ml; the six concordant patients for the two tests had a rate lower than 30 g/100 ml. For the two remaining patients, with an indeterminate 4-RIBA test, serum immunoglobulin levels were 25 and 15 g/100 ml respectively. We
Digestive Diseases and Sciences, Vol. 37, No. 1 (January 1992>
think that the difference observed in the prevalence of anti-HCV antibodies in our series compared to others could be explained, on the one hand, by false-positive ELISA results due to a nonspecific IgG component adhering to the ELISA plates and, on the other, by testing fresh serum samples in this study. Since indeed, it has been suggested that frozen stored serum may give false-positive reactions in an enzyme immunoassay for c100-3 antibody (6). In conclusion, the rate of false-positive first-generation ELISA tests could be estimated in the range of 20-40% in patients with alcoholic liver diseases. As previously observed, this result is closely correlated to serum immunoglobulin concentration of these patients. The HCV-RNA detection after amplification in sera or in hepatic tissues should be helpful in some indeterminate cases. P. LAURENT-PUIG, MD E. DUSSAIX, MD Y. LECOZ, MD P. MA~TES, MD C. BUFFET, MD Service des maladies du foie et de l'appareil digestif Laboratoire de virologie H6pital de Bic~tre 78 Avenue du Gdndral Leclerc 94270 Le Kremlin Bic~tre, France REFERENCES 1. Pares A, Barrera JM, Caballeria J, et al: Hepatitis C virus antibodies in chronic alcoholic patients: Association with severity of liver injury. Hepatology 12:1295-1299, 1990 2. Jacyna MR, Goldin R, McSween R, Thomas HC: Significance of anti-HCV antibodies with alcoholic liver disease from England and Scotland. Gastroenterology 100:A756, 1990 3. Btuix J, Barrera JM, Cavet X, Ercilla G, Costa J, SanchezTapias JM, Ventura M, Vail M, Bruguera M, Bru C, Castillo R, Rodes J: Prevalence of antibodies to hepatitis C virus in a Spanish patient with hepatocellular carcinoma and hepatitis cirrhosis. Lancet 2:1004-1008, 1989 4. Gray JJ, Wreghitt TG, Friend PJ, Wight DGD, Sundaresan V, Calne RY: Differentiation between specific and nonspecific hepatitis C antibodies in chronic liver disease. Lancet 335:610-619, 1990 5. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R: Hepatitis C virus antibodies in chronic active hepatitis: Pathogenic factor or false-positive result. Lancet 335:754-757, 1990 6. Mortimer PP, Cohen BJ, Litton PA, Vandervelde EM, Bassendine MF, Brind AM, Hambling MH: Hepatitis C virus antibody. Lancet 2:798, 1989
157
