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MICROBIAL DEGRADATION OF CARBOXYMETHYLCELLULOSE FROM TAMPONS
279
MICROBIAL DEGRADATION OF
CARBOXYMETHYLCELLULOSE FROM TAMPONS aureus with toxic shock well since first described in documented has been syndrome (TSS) 1978.1 Current evidence suggests that one or more toxins elaborated by strains of S. aureus may be a cause of symptoms in TSS. The striking rise in the incidence of TSS has been ascribed to an as yet unidentified risk factor associated with the use of tampons, particularly the ’Rely’ brand (Procter and Gamble).4,5 A carboxymethylcellulose component of Rely tampons (R-CMC), previously thought to be inert and insoluble, has recently been shown to be liquefiable by the cellulase activity of certain Enterobacteriaceae.6Additional studies (Tierno PM, personal communication) have indicated that certain strains of bacteria can produce reducing sugars from R-CMC, and provide further evidence of its enzymic hydrolysis.
SIR,-The association of Staphylococcus
CELLULASE ACTIVITY OF TEST BACTERIA
particularly klebsiella, escherichia, and proteus, to liquify and release reducing sugars from R-CMC provides further evidence that this component of Rely tampons can be a substrate for bacteria. The discovery that these same microorganisms can be found in substantial numbers in the vagina at different stages of the menstrual cycle9suggests that microbial breakdown of R-CMC can occur in vivo. Department of Pathology, Veterans Administration Medical Center, New York University School of Medicine,
New York, N.Y. 11010, U.S.A.
BRUCE A. HANNA
FACTOR VIII CONCENTRATE: WHAT THE LABEL SAYS, AND STANDARDISATION
SIR,-Dr Austen and colleagues (Nov. 21, p. 1167) and Dr Barrowcliffe and Dr Thomas (Dec. 12, p. 1342) have drawn attention to discrepancies in estimates of potency of factor VIII (FVIII) concentrates. One of us (M. J. S.) has analysed by quantitative elecrophoresis1 and radioimmunoelectrophoresis2all the clinical FVIII concentrates available in the U.K. There was considerable heterogeneity of the concentrates in their multimeric forms3 of FVIII. This heterogeneity may partly explain the discrepancies described by your correspondents; it may also have important clinical implications. An FVIII assay of a concentrate having one pattern ofmultimeric3 forms against a standard having a different pattern (i.e., the international standard) is not strictly a comparison of like with like. In addition, the two-stage FVIII assay is particularly sensitive to the smaller forms of FVIII1,4,5 found in a greater proportion in manufacturer A’s preparations than in other concentrates, including the international standard. There are thus at least two possible technical reasons for discrepancies between estimates of concentrate potency. On the clinical side, it appears that in vivo recovery, or the half life, of the smaller forms, is less than that for the clinically more active larger forms.This may account for the lack of clinical efficacy of some concentrates as reported by Austen et al. The smaller forms of FVIII also lack the components required for the effective treatment of patients with von Willebrand disease. The origin of the smaller forms in the concentrates is unknown, but it may be due
to selective enrichment by certain production procedures and/or generated by partial proteolysis of larger multimers.
We suggest that technical aspects of the assays used and the I
I
I
I
To measure the extent to which cellulase activity might be present representative microorganisms, we tested sixty bacterial isolates by a modified viscosimetric method with R-CMC as of these same isolates to release substrate,6 and examined the ability reducing sugars from R-CMC.7 Our results (table) indicate that cellulase activity is detectable not only in Enterobacteriaceae but also in Pseudomonadaceae and in members of the genus Achromobacter. Since cellulase complexes can be resolved into distinct component parts, each of which attacks the substrate at a specific site,8 the ability of many strains of bacteria,
among
1 Todd J, Fishaut M, Kapral F, Welch T Toxic shock syndrome associated with group-1
staphylococci. Lancet 1978; ii: 1116-18. 2. Schlievert PM, Shands KN, Dan BB, Schmid GP, Nishimura RD. Identification and characterization of an exotoxin from Staphylococcus aureus associated with toxicshock syndrome. J Infect Dis 1981; 143: 509-16. 3 Bergdoll MS, Reiser RF, Cross BA, Robbins RN, Davis JP A new staphylococcal enterotoxin, enterotoxin F, associated with toxic-shock syndrome Sraphylococcus aureus isolates Lancet 1981; i: 1017-21. 4 Centers for Disease Control. Follow-up on toxic-shock-United States. Morbid Mortal Weekly Rep 1980, 29: 297-99. 5 Centers for Disease Control. TSS-Utah. Morbid Mortal Weekly Rep 1980; 29: 495-96. 6. Tierno PM. Cellulase activity of microorganisms on carboxymethylcellulose from tampons Lancet 1981; ii: 746-47. 7. Mandels M, Andreotti R, Roche C. Measurement of saccharifying cellulase Biotech Bioeng Symp 1976; 6: 21-33. 8. Wood TM Properties and mode of action of cellulases. Biotech Bioeng Symp 1975; 5: 111-37
varying functional activities of different multimeric forms need to be taken into account in estimating the potency of FVIII concentrates. Recognition of these points is likely to help not only in clarifying the reasons for discrepancies in assay results but also in the management of patients with FVIII disorders. North London Blood Transfusion Centre, Edgware, Middlesex
M. J. SEGHATCHIAN
Epidemiology and Medical Care Unit, Northwick Park Hospital, Harrow, Middlesex
YVONNE STIRLING
9. Botta GA, Pedulla D, Melioli G, MadoffS, Minuto F. Absence of
fluctuation in vaginal by Enterobacteriaceae during the menstrual cycle in patients with recurrent cystitis. Lancet 1981; i: 1116-17. 1. Seghatchian MJ. An agarose gel method for evaluating FVII procoagulant electrophoretic distribution. Ann NY Acad Sci 1981; 370: 236-40. 2. Seghatchian MJ, Mackie IJ. FVIII coagulant antigen in clinical factor IX concentrates: colonisation
Characterisation of the molecular forms with the use of radiolabelled FVIII:C antibody. Thrombosis Res 1982; 24: 33-38. 3. Ruggeri ZM, Zimmerman TS. Multimeric composition of FVIII. Ann NY Acad Sci 1981. 370: 205-09. 4. Seghatchian MJ, Mackie IJ. Aluminium hydroxide absorption and FVIII clotting assays. Lancet 1980; ii: 38. 5. Seghatchian MJ, Hirst K. Evidence for the presence of FVIII subunits in plasma and clinical FVIII concentrates using quantitative electrophoresis. Proc Int Congr Thromb Haemostas 1979; 42: 65 (abstr). 6. Nilsson IM, Holmberg C, Stenberg P, Henrickss P. Characterisation of FVIII protein and FXIII in various FVIII concentrates. Scand J Haematol 1980; 24: 340-49. 7. Lombardi R, Mannucci PM. Seghatchian MJ, Vicente Garcia V, Coppola R. Alteration of FVIII von Willebrand factor in clinical conditions associated with an increase in its plasma concentration. Br J Haematol 1981; 49: 61-68.
,
MICROBIAL DEGRADATION OF
CARBOXYMETHYLCELLULOSE FROM TAMPONS aureus with toxic shock well since first described in documented has been syndrome (TSS) 1978.1 Current evidence suggests that one or more toxins elaborated by strains of S. aureus may be a cause of symptoms in TSS. The striking rise in the incidence of TSS has been ascribed to an as yet unidentified risk factor associated with the use of tampons, particularly the ’Rely’ brand (Procter and Gamble).4,5 A carboxymethylcellulose component of Rely tampons (R-CMC), previously thought to be inert and insoluble, has recently been shown to be liquefiable by the cellulase activity of certain Enterobacteriaceae.6Additional studies (Tierno PM, personal communication) have indicated that certain strains of bacteria can produce reducing sugars from R-CMC, and provide further evidence of its enzymic hydrolysis.
SIR,-The association of Staphylococcus
CELLULASE ACTIVITY OF TEST BACTERIA
particularly klebsiella, escherichia, and proteus, to liquify and release reducing sugars from R-CMC provides further evidence that this component of Rely tampons can be a substrate for bacteria. The discovery that these same microorganisms can be found in substantial numbers in the vagina at different stages of the menstrual cycle9suggests that microbial breakdown of R-CMC can occur in vivo. Department of Pathology, Veterans Administration Medical Center, New York University School of Medicine,
New York, N.Y. 11010, U.S.A.
BRUCE A. HANNA
FACTOR VIII CONCENTRATE: WHAT THE LABEL SAYS, AND STANDARDISATION
SIR,-Dr Austen and colleagues (Nov. 21, p. 1167) and Dr Barrowcliffe and Dr Thomas (Dec. 12, p. 1342) have drawn attention to discrepancies in estimates of potency of factor VIII (FVIII) concentrates. One of us (M. J. S.) has analysed by quantitative elecrophoresis1 and radioimmunoelectrophoresis2all the clinical FVIII concentrates available in the U.K. There was considerable heterogeneity of the concentrates in their multimeric forms3 of FVIII. This heterogeneity may partly explain the discrepancies described by your correspondents; it may also have important clinical implications. An FVIII assay of a concentrate having one pattern ofmultimeric3 forms against a standard having a different pattern (i.e., the international standard) is not strictly a comparison of like with like. In addition, the two-stage FVIII assay is particularly sensitive to the smaller forms of FVIII1,4,5 found in a greater proportion in manufacturer A’s preparations than in other concentrates, including the international standard. There are thus at least two possible technical reasons for discrepancies between estimates of concentrate potency. On the clinical side, it appears that in vivo recovery, or the half life, of the smaller forms, is less than that for the clinically more active larger forms.This may account for the lack of clinical efficacy of some concentrates as reported by Austen et al. The smaller forms of FVIII also lack the components required for the effective treatment of patients with von Willebrand disease. The origin of the smaller forms in the concentrates is unknown, but it may be due
to selective enrichment by certain production procedures and/or generated by partial proteolysis of larger multimers.
We suggest that technical aspects of the assays used and the I
I
I
I
To measure the extent to which cellulase activity might be present representative microorganisms, we tested sixty bacterial isolates by a modified viscosimetric method with R-CMC as of these same isolates to release substrate,6 and examined the ability reducing sugars from R-CMC.7 Our results (table) indicate that cellulase activity is detectable not only in Enterobacteriaceae but also in Pseudomonadaceae and in members of the genus Achromobacter. Since cellulase complexes can be resolved into distinct component parts, each of which attacks the substrate at a specific site,8 the ability of many strains of bacteria,
among
1 Todd J, Fishaut M, Kapral F, Welch T Toxic shock syndrome associated with group-1
staphylococci. Lancet 1978; ii: 1116-18. 2. Schlievert PM, Shands KN, Dan BB, Schmid GP, Nishimura RD. Identification and characterization of an exotoxin from Staphylococcus aureus associated with toxicshock syndrome. J Infect Dis 1981; 143: 509-16. 3 Bergdoll MS, Reiser RF, Cross BA, Robbins RN, Davis JP A new staphylococcal enterotoxin, enterotoxin F, associated with toxic-shock syndrome Sraphylococcus aureus isolates Lancet 1981; i: 1017-21. 4 Centers for Disease Control. Follow-up on toxic-shock-United States. Morbid Mortal Weekly Rep 1980, 29: 297-99. 5 Centers for Disease Control. TSS-Utah. Morbid Mortal Weekly Rep 1980; 29: 495-96. 6. Tierno PM. Cellulase activity of microorganisms on carboxymethylcellulose from tampons Lancet 1981; ii: 746-47. 7. Mandels M, Andreotti R, Roche C. Measurement of saccharifying cellulase Biotech Bioeng Symp 1976; 6: 21-33. 8. Wood TM Properties and mode of action of cellulases. Biotech Bioeng Symp 1975; 5: 111-37
varying functional activities of different multimeric forms need to be taken into account in estimating the potency of FVIII concentrates. Recognition of these points is likely to help not only in clarifying the reasons for discrepancies in assay results but also in the management of patients with FVIII disorders. North London Blood Transfusion Centre, Edgware, Middlesex
M. J. SEGHATCHIAN
Epidemiology and Medical Care Unit, Northwick Park Hospital, Harrow, Middlesex
YVONNE STIRLING
9. Botta GA, Pedulla D, Melioli G, MadoffS, Minuto F. Absence of
fluctuation in vaginal by Enterobacteriaceae during the menstrual cycle in patients with recurrent cystitis. Lancet 1981; i: 1116-17. 1. Seghatchian MJ. An agarose gel method for evaluating FVII procoagulant electrophoretic distribution. Ann NY Acad Sci 1981; 370: 236-40. 2. Seghatchian MJ, Mackie IJ. FVIII coagulant antigen in clinical factor IX concentrates: colonisation
Characterisation of the molecular forms with the use of radiolabelled FVIII:C antibody. Thrombosis Res 1982; 24: 33-38. 3. Ruggeri ZM, Zimmerman TS. Multimeric composition of FVIII. Ann NY Acad Sci 1981. 370: 205-09. 4. Seghatchian MJ, Mackie IJ. Aluminium hydroxide absorption and FVIII clotting assays. Lancet 1980; ii: 38. 5. Seghatchian MJ, Hirst K. Evidence for the presence of FVIII subunits in plasma and clinical FVIII concentrates using quantitative electrophoresis. Proc Int Congr Thromb Haemostas 1979; 42: 65 (abstr). 6. Nilsson IM, Holmberg C, Stenberg P, Henrickss P. Characterisation of FVIII protein and FXIII in various FVIII concentrates. Scand J Haematol 1980; 24: 340-49. 7. Lombardi R, Mannucci PM. Seghatchian MJ, Vicente Garcia V, Coppola R. Alteration of FVIII von Willebrand factor in clinical conditions associated with an increase in its plasma concentration. Br J Haematol 1981; 49: 61-68.
,