- Email: [email protected]
LYSINOALANINE CONTENTS OF FORMULA DIETS
448 CMV being positive at titres 40 on day 100 of life (3 weeks after he became acutely ill), 80 on day 126, and 160 on day 138. He was discharged home on vitamin supplements alone, aged 20 weeks. This infant acquired a severe CMV infection whilst in a neonatal nursery. Serological tests showed that this was not a congenital CMV infection and that the infection was not due to other respiratory viruses. Culture of CMV from bronchial secretions but never from pharyngeal secretions supports the fact that he had a primary CMV pneumonia. Transfusion carries a risk of CMV infection that is thought to be proportional to the number of units transfused and estimated as being between 5% and 12% per unit.4,5 This infant improved clinically only after the use of hyperimmune plasma. Earlier reports in renal transplantpatients have been encouraging,3and this treatment for severe neonatal CMV infection should be assessed in other centres. Department of Child Health, University of Liverpool, M. J. SMITH Liverpool Maternity Hospital, R. W. I. COOKE Liverpool L7 7BN N. HOOD and Department of Medical Microbiology, C. A. HART University of Liverpool Blood Transfusion Service, Edinburgh, SE Scotland
P. L. YAP
LYSINOALANINE CONTENTS OF FORMULA DIETS
SIR,-Lysinoalanine (LAL: N-E[D,L-2-amino-2-carboxyethyl]L-lysine) is a cross-linking aminoacid which may be found in processed proteinaceous foods after alkali and heat treatment.6 It is formed by a reaction of a dehydroalanine residue (from cysteine/cystine or serine) with the E-amino group of lysine. Free LAL is toxic in rats, with developing in animals on diets containing LAL;7, no renal changes were encountered, however, in mice, hamsters, rabbits, quail, dogs, or Feeding experiments in laboratory animals9and tests of in vitro show that protein containing bound LAL is of inferior nutritional quality. Bound LAL is of low toxicity in rats. Although the importance of LAL in terms of human toxicity
nephrocytomegaly
monkeys.s
digestibility’ 0, "
unclearl2 it is generally accepted that with a high of LAL is of reduced nutritional 3 partly because of loss of the essential aminoacids cysteine/cystine and lysine and partly because of the reduced digestibility of cross-linked protein chains. Unwanted effects are most likely with diets where LALdamaged protein is the sole or at least dominant source of protein. This applies, for example, to formula diets administered to patients in a reduced metabolic state (eg, in critical care medicine, remains
content
value,protein
gastroenterology, -and oncology). In a survey of formula diets available in Germany, the bound LAL of fifteen products (ten powder five ready-toIn powders no LAL was use liquid formulae) was determined. detectable (<20 ppm LAL in the protein or <20 laglg), with one exception-a sample containing 120 ppm, still an acceptable content
preparations,
4. Ho M. Cytomegalovirus. In: Mandell GL, Douglas RG, Bennett JG, eds. Principles and practice of infectious diseases. New York: Wiley, 1979: 1307-323. 5. Benson JWT, Bodden SJ, Tobin JO’H. Cytomegalovirus and blood transfusion in neonates. Arch Dis Child 1979; 54: 538-41. 6. Friedman M, ed. Protein crosslinking: Nutritional and medical consequences. (Adv Exp Med Biol 86B). New York: Plenum, 1977. 7. Woodward JC, Short DD. Toxicity of alkali-treated soyprotein in rats. J Nutr 1973;
103: 569-74.
AP, Slump P, Feron VJ, van Beek L. Effects of alkali-treated proteins: Feeding studies with free and protein-bound lysinoalanine in rats and other animals. J Nutr 1976; 106: 1527-38. 9. Struthers BJ, Hopkins DT, Prescher EE, Dahlgren RR. Effects of protein-bound lysinoalanine, N-∈(DL-2-amino-2-carboxyethyl)-L-lysine, on fetal and neonatal rats. J Nutr 1978; 108: 954-58. 10. Friedman M, Zahnley JC, Masters PM. Relationship between m vitro digestibility of casein and its content of lysinoalanine and D-amino acids. J Food Sci 1981; 462: 8. De Groot
127-31,34. C, Gauthier SF, Jones JD, Savoie L. Ezymatic digestion method with dialysis to assess protein damage: Application to alkali-treated proteins containing lysinoalanine. Nutr Res 1982; 2: 675-88.
11. Vachon
12. Pfaender P. Lysinoalanine: A toxic compound in processed proteinaceous foods. Wld Rev Nutr Diet 1983; 41: 97-109. 13. Satterlee LD, Chang KC. Nutritional quality of deteriorated proteins. In: Cherry JP, ed. Food protein deterioration (ACS Symp Series 206). Washington: American Chemical Society, 1982; 409-31. 14. Fritsch RJ, Klostermeyer H. Empfindliche Bestimmung von Lysinoalanin und 2,3-Diaminopropionsäure durch fluorimetrische Detektion mit orthoPhtaldialdehyd. Z Lebensm UntersForsch 1981; 173: 101-06.
concentration. The liquid preparations contained 485, 1000, 1160, 1430, and 1730 ppm. In comparison with the LAL content of processed foods6,12,is these levels are disconcertingly high and seem to indicate definite protein damage. In a Netherlands draft regulation on protein products 16 suggested a limit of 200 ppm for bound LAL. All the liquid preparations analysed were above this limit. Lysinoalanine is only one of several indicators of protein damage and, therefore, reduced nutritional value: other crosslinking aminoacids, such as ornithinoalanine and lanthionine, Maillard products, and racemised D-aminoacids were not measured.13 The formation of LAL in liquid formulae seems to be linked to intense, perhaps repeated, heat treatment during processing and sterilisation; it is also likely that LAL-damaged protein isolates were used in the manufacturing process. These technical problems can probably be overcome by the use of careful processing techniques. The adverse effects of LAL-damaged protein in formula diets should be further evaluated, since this is theoretically a factor limiting nitrogen absorption and nitrogen balance in patients with a reduced metabolic state. I suggest the use of protein preparations containing less than 200 ppm LAL, and preferably containing no detectable LAL. This investigation gemeinschaft.
was
supported by
the
Deutsche
Department of Paediatric Surgery, University of Heidelberg, D-6900 Heidelberg, West Germany
Forschungs-
GÜNTER PFAFF
PRODUCTION OF CHOLERA-LIKE TOXIN BY
CAMPYLOBACTER JEJUNI/COLI SIR,-Work in our laboratory supports the conclusion of RuizPalacios and colleagues that isolates of Campylobacter jejuni produce a cholera-like enterotoxin. C jejunilcoli clinical, food, and animal isolates were grown overnight in casein/aminoacid yeast extract broth,2brucella broth (Difco), or brucella broth containing lincomycin at 37°C with shaking in a static atmosphere of 85% nitrogen, 10% carbon dioxide, and 5% oxygen. No growth supplement or blood was added to
the media. Final cell concentrations
were
between 5 x 108 and ’
5 x 109 colony forming units/ml. Cell-free supernatants from seven of eight clinical, one of two animal, and one of two food isolates were positive (absorbance at 4100
(ELISA)3
nm 0-1) in an enzyme-linked immunosorbent assay based on binding of toxin to cholera toxin (CT) antibody. In tests for enterotoxins filter-sterilised (0-2jum, Gelman, Ann Arbor,
Michigan) five-fold concentrates ofC jejunilcoli supernatants caused rounding of Y-1 mouse adrenal cells and elongation of Chinese hamster ovary cells. Rounding of Y-1 mouse adrenal cells was eliminated by preincubation of the campylobacter toxin with antiCT antibody for 1 h at 37° C. Twenty-fold supernatant concentrates of C jejuni strain CH5, a human faecal isolate, caused fluid accumulation in rabbit ileal loops and increased permeability in the rabbit skin test. The campylobacter toxin
was eluted from an anti-CT affinity column4in a single peak by "tris" buffer, pH 10-5. Upon sodium dodecylsulphate gel electrophoresis, the eluted material produced one band. Antibody was produced by injection of the affinity column-purified toxin with Freund’s incomplete adjuvant into rabbits over a period of several weeks. Antibody to CT (Sigma Chemical) was prepared in a similar manner. IgG was isolated from both CT and campylobacter antisera on a protein A ’Sepharose’
15. 16.
Steinig J, Montag A. Studien über Veränderungen des Lysins im Nahrungsprotein. Tell II. Bildung von Lysinoalanin. Z Lebensm Unters Forsch 1982; 175: 8-12. Anon. Ontwerp-Eiwitproduktenbesluit(Warenwet). Nederlandse Staatscourant Feb 14,
1979, no 32. 1. Ruiz-Palacios GM, Torres
J, Torres N, et al. Cholera-like enterotoxin produced by Campylobacter Jejuni. Lancet 1983; ii: 250-52. 2. Kusama H, Craig JP. Production of biologically active substances by two strains of Vibrio cholerae. Infect Immun 1970; 1: 80-87. 3. Yolken RH, Greenberg HB, Merson MH, Sack RB, Kapikian AZ. Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin. J Clin Microbiol 1977; 6: 439-44. Z, Robbin JB. Purification of heat-labile enterotoxin from Escherichia coli 078:H11by affinity chromatography with antisera to Vibrio cholerae toxin. J Infect Dis 1976; 133 (suppl): S138-S141.
4. Dafni
P. L. YAP
LYSINOALANINE CONTENTS OF FORMULA DIETS
SIR,-Lysinoalanine (LAL: N-E[D,L-2-amino-2-carboxyethyl]L-lysine) is a cross-linking aminoacid which may be found in processed proteinaceous foods after alkali and heat treatment.6 It is formed by a reaction of a dehydroalanine residue (from cysteine/cystine or serine) with the E-amino group of lysine. Free LAL is toxic in rats, with developing in animals on diets containing LAL;7, no renal changes were encountered, however, in mice, hamsters, rabbits, quail, dogs, or Feeding experiments in laboratory animals9and tests of in vitro show that protein containing bound LAL is of inferior nutritional quality. Bound LAL is of low toxicity in rats. Although the importance of LAL in terms of human toxicity
nephrocytomegaly
monkeys.s
digestibility’ 0, "
unclearl2 it is generally accepted that with a high of LAL is of reduced nutritional 3 partly because of loss of the essential aminoacids cysteine/cystine and lysine and partly because of the reduced digestibility of cross-linked protein chains. Unwanted effects are most likely with diets where LALdamaged protein is the sole or at least dominant source of protein. This applies, for example, to formula diets administered to patients in a reduced metabolic state (eg, in critical care medicine, remains
content
value,protein
gastroenterology, -and oncology). In a survey of formula diets available in Germany, the bound LAL of fifteen products (ten powder five ready-toIn powders no LAL was use liquid formulae) was determined. detectable (<20 ppm LAL in the protein or <20 laglg), with one exception-a sample containing 120 ppm, still an acceptable content
preparations,
4. Ho M. Cytomegalovirus. In: Mandell GL, Douglas RG, Bennett JG, eds. Principles and practice of infectious diseases. New York: Wiley, 1979: 1307-323. 5. Benson JWT, Bodden SJ, Tobin JO’H. Cytomegalovirus and blood transfusion in neonates. Arch Dis Child 1979; 54: 538-41. 6. Friedman M, ed. Protein crosslinking: Nutritional and medical consequences. (Adv Exp Med Biol 86B). New York: Plenum, 1977. 7. Woodward JC, Short DD. Toxicity of alkali-treated soyprotein in rats. J Nutr 1973;
103: 569-74.
AP, Slump P, Feron VJ, van Beek L. Effects of alkali-treated proteins: Feeding studies with free and protein-bound lysinoalanine in rats and other animals. J Nutr 1976; 106: 1527-38. 9. Struthers BJ, Hopkins DT, Prescher EE, Dahlgren RR. Effects of protein-bound lysinoalanine, N-∈(DL-2-amino-2-carboxyethyl)-L-lysine, on fetal and neonatal rats. J Nutr 1978; 108: 954-58. 10. Friedman M, Zahnley JC, Masters PM. Relationship between m vitro digestibility of casein and its content of lysinoalanine and D-amino acids. J Food Sci 1981; 462: 8. De Groot
127-31,34. C, Gauthier SF, Jones JD, Savoie L. Ezymatic digestion method with dialysis to assess protein damage: Application to alkali-treated proteins containing lysinoalanine. Nutr Res 1982; 2: 675-88.
11. Vachon
12. Pfaender P. Lysinoalanine: A toxic compound in processed proteinaceous foods. Wld Rev Nutr Diet 1983; 41: 97-109. 13. Satterlee LD, Chang KC. Nutritional quality of deteriorated proteins. In: Cherry JP, ed. Food protein deterioration (ACS Symp Series 206). Washington: American Chemical Society, 1982; 409-31. 14. Fritsch RJ, Klostermeyer H. Empfindliche Bestimmung von Lysinoalanin und 2,3-Diaminopropionsäure durch fluorimetrische Detektion mit orthoPhtaldialdehyd. Z Lebensm UntersForsch 1981; 173: 101-06.
concentration. The liquid preparations contained 485, 1000, 1160, 1430, and 1730 ppm. In comparison with the LAL content of processed foods6,12,is these levels are disconcertingly high and seem to indicate definite protein damage. In a Netherlands draft regulation on protein products 16 suggested a limit of 200 ppm for bound LAL. All the liquid preparations analysed were above this limit. Lysinoalanine is only one of several indicators of protein damage and, therefore, reduced nutritional value: other crosslinking aminoacids, such as ornithinoalanine and lanthionine, Maillard products, and racemised D-aminoacids were not measured.13 The formation of LAL in liquid formulae seems to be linked to intense, perhaps repeated, heat treatment during processing and sterilisation; it is also likely that LAL-damaged protein isolates were used in the manufacturing process. These technical problems can probably be overcome by the use of careful processing techniques. The adverse effects of LAL-damaged protein in formula diets should be further evaluated, since this is theoretically a factor limiting nitrogen absorption and nitrogen balance in patients with a reduced metabolic state. I suggest the use of protein preparations containing less than 200 ppm LAL, and preferably containing no detectable LAL. This investigation gemeinschaft.
was
supported by
the
Deutsche
Department of Paediatric Surgery, University of Heidelberg, D-6900 Heidelberg, West Germany
Forschungs-
GÜNTER PFAFF
PRODUCTION OF CHOLERA-LIKE TOXIN BY
CAMPYLOBACTER JEJUNI/COLI SIR,-Work in our laboratory supports the conclusion of RuizPalacios and colleagues that isolates of Campylobacter jejuni produce a cholera-like enterotoxin. C jejunilcoli clinical, food, and animal isolates were grown overnight in casein/aminoacid yeast extract broth,2brucella broth (Difco), or brucella broth containing lincomycin at 37°C with shaking in a static atmosphere of 85% nitrogen, 10% carbon dioxide, and 5% oxygen. No growth supplement or blood was added to
the media. Final cell concentrations
were
between 5 x 108 and ’
5 x 109 colony forming units/ml. Cell-free supernatants from seven of eight clinical, one of two animal, and one of two food isolates were positive (absorbance at 4100
(ELISA)3
nm 0-1) in an enzyme-linked immunosorbent assay based on binding of toxin to cholera toxin (CT) antibody. In tests for enterotoxins filter-sterilised (0-2jum, Gelman, Ann Arbor,
Michigan) five-fold concentrates ofC jejunilcoli supernatants caused rounding of Y-1 mouse adrenal cells and elongation of Chinese hamster ovary cells. Rounding of Y-1 mouse adrenal cells was eliminated by preincubation of the campylobacter toxin with antiCT antibody for 1 h at 37° C. Twenty-fold supernatant concentrates of C jejuni strain CH5, a human faecal isolate, caused fluid accumulation in rabbit ileal loops and increased permeability in the rabbit skin test. The campylobacter toxin
was eluted from an anti-CT affinity column4in a single peak by "tris" buffer, pH 10-5. Upon sodium dodecylsulphate gel electrophoresis, the eluted material produced one band. Antibody was produced by injection of the affinity column-purified toxin with Freund’s incomplete adjuvant into rabbits over a period of several weeks. Antibody to CT (Sigma Chemical) was prepared in a similar manner. IgG was isolated from both CT and campylobacter antisera on a protein A ’Sepharose’
15. 16.
Steinig J, Montag A. Studien über Veränderungen des Lysins im Nahrungsprotein. Tell II. Bildung von Lysinoalanin. Z Lebensm Unters Forsch 1982; 175: 8-12. Anon. Ontwerp-Eiwitproduktenbesluit(Warenwet). Nederlandse Staatscourant Feb 14,
1979, no 32. 1. Ruiz-Palacios GM, Torres
J, Torres N, et al. Cholera-like enterotoxin produced by Campylobacter Jejuni. Lancet 1983; ii: 250-52. 2. Kusama H, Craig JP. Production of biologically active substances by two strains of Vibrio cholerae. Infect Immun 1970; 1: 80-87. 3. Yolken RH, Greenberg HB, Merson MH, Sack RB, Kapikian AZ. Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin. J Clin Microbiol 1977; 6: 439-44. Z, Robbin JB. Purification of heat-labile enterotoxin from Escherichia coli 078:H11by affinity chromatography with antisera to Vibrio cholerae toxin. J Infect Dis 1976; 133 (suppl): S138-S141.
4. Dafni