192
mental system. It is based on the old observation that under suitable conditions the interaction of cysteine, iron, and atmospheric oxygen leads to the formation of a transient coloured complex. Mainly by virtue of its nitro group, metronidazole can take the place of oxygen in this reaction, the cysteine/ iron/drug complex decaying through free-radical intermediates. The inhibitory effect of zinc on the oxygen consumption of the cysteine/iron mixture was described in 1909 by MATHEWS and WALKER.16 More recent studies by WILLSON have shown that zinc may also inhibit the analogous metronidazoledependent free-radical sequence.4,17 Very little can be taken for granted with either trace elements or free radicals. After several decades of zinc studies in the laboratory the first human disease which can be specifically attributed to zinc deficiency came as a surprise to most, perhaps all, workers in the field.l8 Acrodermatitis enteropathica is a rare inborn metabolic disorder; and how exactly the zinc deficiency arises in the patients is still uncertain. But at a highly speculative level it is perhaps relevant that the disease affects a tissue which is most continuously exposed to ultraviolet irradiation. (In the same speculative vein one may note that of all human organs it is the eye which has the highest zinc content; and that seminal fluid, the body fluid with the highest zinc concentration, is the only one in which cells must survive outside the body if they are to fulfil their
physiological function.) Zinc may prove
be rather less prominent in protecting against iron-catalysed free-radical dammage than has been suggested, but if the suggestion catalyses a "radical" approach to pathological and medical problems it will have served a useful purpose. Such an approach might prove the more opportune in the light of recent methodological advances. Most free radicals are far too short-lived to be detectable in tissues (although free-radical signals have been recorded in cataractous lensesI9); and even the primary products of free-radical reactions tend to be unstable. For several decades almost the only chemical "marker" of biological free-radical activity has been malonyldialdehyde or M.D.A., a secondary breakdown product of polyunsaturated lipids destroyed by free-radical oxidation.2o,21 (In fact, M.D.A. is only one of many freeradical-oxidation products which give the same characteristic colour reaction with thiobarbituric acid: hence the alternative term of T.B.A.-reactive compounds.22) It has been a useful tool but it has severe limitations, especially when applied to to
16. Mathews, A. P., Walker, S. J. biol. Chem. 1909, 6, 312. 17. Willson, R. L. Lancet, 1976, i, 1407. 18. Moynahan, E. J. ibid. 1974, i, 399. 19. Weiter, J. J., Finch, E. D. Nature, 1975, 254, 536. 20. Kohn, H. I., Liversedge, M. J. Pharmacol. 1944, 82, 292. 21. Stocks, J., Dormandy, T. L. Br. J. Hœmat. 1971, 20, 95. 22. Gutteridge, J. M. C., Stocks, J., Dormandy, T. L. Anal. chim. 70, 107.
Acta, 1974,
human pathology. More recent work suggests that fluorescence spectroscopy may provide a better insight into in-vivo free-radical processes.23 The fluorescent spectra of complex biological mixtures are often difficult to interpret and to quantitate; but the difficulties may prove to be a challenge rather than a deterrent.
Treatment of
Chlamydia trachomatis
Infections Chlamydia trachomatis are common. Hyperendemic trachoma affects an estimated 500 million people in developing countries.’ In England and Wales, upwards of 70 000 cases of non-gonococcal urethritis (N.G.u.) are reported annually, of which about half will be caused by INFECTIONS with
C. trachomatis.2,3 To these must be added paratrachoma,ophthalmia neonatorum,S pneumonitis of
infants,6 salpingitis,7 "non-specific" genital infection in women,8 and post-gonococcal urethritis.9 There is now considerable evidence that the cervix of female contacts of men with N.G.U. forms a substantial reservoir of C trachomatis,10 whilst the incidence of concurrent C. trachomatis infection in women with gonococcal cervicitis may be as high as 62%." The diversity and scale of chlamydial infections has stimulated laboratory studies initially to simplify isolation of the organism, and more recently to investigate the in-vitro effects of antimicrobial agents against C. trachomatis, with a view to rationalising chemotherapy. Early in-vitro work was done with the yolk-sac of embryonated hen’s eggs.12 This technique is cumbersome and not suitable for rapid screening, either of antimicrobials or of large numbers of C. trachomatis isolates. Cell-culture provides a more convenient way of investigating the action of antimicrobials on this organism. RIDGWAY, OWEN, and ORIEL" examined the activity of eight antimicrobials on a single strain of C. trachomatis. The most active compounds were erythromycin and oxytetra23. Dillard, C. J., Tappel, A. K. Lipids, 1973, 8, 183. 1. Lancet, 1977, ii, 857. 2. Holmes, K. K., et al. New Engl. J. Med. 1975, 292, 1199. 3. Oriel, J. D., Reeve, P., Wright, J. T., Owen, J. Br. J. vener. Dis. 1976, 52, 46. 4. Darougar, S., Jones, B. R., Kinnison, J. R., Vaughan Jackson, T. D., Dunlop, E. ibid. 1972, 48, 416. 5. Rees, E., Tait, I. A., Hobson, D., Byng, R. E., Johnson, F. W. A. ibid. 1977,
53, 173. 6. Beem, M. O., Saxon, E. M. New Engl. J. Med. 1977, 296, 306. 7. Mårdh, P. A., Ripa, T., Svensson, L., Weström, L. ibid. 1977, 296, 1377. 8. Rees, E., Tait, I. A., Hobson, D., Johnson, F. W. A. in Non Gonococcal Urethritis and Related Infections (edited by D. Hobson and K. K. Holmes); p. 67. American Society for Microbiology, Washington, D.C., 1977. 9. Oriel, J. D., Reeve, P., Thomas, B. J., Nicol, C. S. J. infect. Dis. 1975, 131, 376. 10. Oriel, J. D., Powis, P. A., Reeve, P., Miller, A., Nicol, C. S. Br. J. vener. Dis. 1974, 50, 11. 11. Hilton, A. L., Richmond, S. J., Milne, J. D., Hindley, F., Clarke, S. K. R. ibid. 1974, 50, 1. 12. Werner, G. H. Ann. Inst. Pasteur, 1961, 100, 93. 13. Ridgway, G. L., Owen, J., Oriel, J. D. J. antimicrob. Chemother. 1976, 2, 71.
193 TREHARNE et al. 14 found
rifampicin more active than erythromycin or oxytetracycline, whilst both groups found sulphonamides, trimethoprim, chloramphenicol, and the penicillins only moderately active. Spectinomycin, metronidazole, and aminoglycosides were inactive. The experience of Kuo, WANG, and GRAYSTON1S was similar: they also noted that rosamicin (a new macrolide antibiotic) was just as active as erythromycin, that different
cycline.
serotypes of C. trachomatis varied in sensitivity to sulphonamides, and that penicillin gave rise to abnormal forms of C. trachomatis. Latency is a well-known phenomenon in C. psittaci infections, and has been suggested as an explanation for some of the clinical findings in oculogenital infections with C. trachomatis.16.17 In vitro, this phenomenon has been seen in C. psittaca’’8 and C. trachomatis.19 Whilst latency was particularly striking with the cell-wall inhibiting antibiotics and the sulphonamides, RIDGWAY et al. 19 found that all antimicrobials tested could induce latency after serial passage. Clearly, caution is required in the interpretation of sensitivity tests with C. trachomatis on single passage. Closely related antimicrobials may have widely differing inhibitory concentrations; thus, results with a single antimicrobial should not be taken to apply to related antimicrobials. Ocular chlamydial infections respond to 5 or 6 weeks’ treatment with tetracycline or rifampicin eye ointment. In 1972, when the role of C. trachomatis in N.G.u. was still in doubt, WILLCOX2o concluded on clinical grounds that ampicillin, nalidixic acid, and novobiocin were no better than placebo; and other compounds of questionable value included sulphonamides, penicillin, chloramphenicol, metronidazole, and spectinomycin. These findings are now borne out by in-vitro tests on C. trachomatis. Tetracyclines, by contrast, have long been known to be effective for N.G.u., and eradication of C. trachomatis coincides with resolution of symptoms.21,22 In patients with gonococcal urethritis ORIEL and his co-workers,9.23,24 showed that coinci dent infection with C. trachomatis was unaffected by gentamicin, ampicillin, and spectinomycin. These patients went on to acquire postgonococcal 14. Treharne, J. D., Day, J., Yeo, C. K., Jones, B. R., Squires, S. in Non Gonococcal Urethritis and Related Infections (edited by D. Hobson and K. K. Holmes); p. 214. American Society for Microbiology, Washington, D.C., 1977. 15. Kuo, C. C., Wang, S. P., Grayston, J. T. Antimicrob. Agents Chemother.
1977, 12, 80. 16. Hannah, L., Dawson, C. R., Briones, O.,
mun. 1968, 101, 43. 17. Richmond, S. J., Hilton,
A. L.,
Clarke,
Thygeson, P., Jawetz,
S. K. Br.
J.
vener.
Dis.
E.
J.
Im-
1972, 48,
437. 18. Hatch, T. P. Infect. Immun. 1975, 12, 211. 19. Ridgway, G. L., Owen, J., Oriel, J. D. Br. J. vener. Dis. (in the press). 20. Willcox, R. R. ibid. 1972, 48, 137. 21. Oriel, J. D., Reeve, P., Nicol, C. S. J. Am. vener. Dis. Ass. 1975, 2, 17. 22. Prentice, M. J., Taylor-Robinson, D., Csonka, G. W. Br. J. vener. Dis. 1976,
52, 269. J. D., Ridgway, G. L., Reeve, P., Beckingham, D. C., Owen, J. J. infect. Dis. 1976, 133, 568. Oriel, J. D., Ridgway, G. L., Tchomouroff, S., Owen, J. Br. J. vener. Dis.
23. Oriel, 24
1977, 53, 226.
urethritis; clearly, when treatment schedules for gonorrhoea are revised, we ought to consider a regimen effective against both Neisseria gonorrhcece and C. trachomatis. Some workers have recommended 3 weeks’ treatment for N.G.U., but it may be unrealistic to expect patients to persevere with medication every six hours for this long, when for much of the time they will be symptom-free. ORIEL et al. 25 found that 250 mg of oxytetracycline sixhourly for 2 weeks gave an 86% cure-rate, as did 500 mg of erythromycin stearate twelve-hourly for 2 weeks. Rifampicin has yet to be evaluated in the treatment of N.G.U. Whether single-dose chemotherapy is feasible (as now practised for gonococcal urethritis) remains to be seen. Treatment with antimicrobials has been employed with some success in the elucidation of other possible agents of N.G.U. HANDSFIELD et a1.26 found that the response of N.G.U. to tetracycline therapy was better in Chlamydia-positive patients than in Chlamydia-negative patients. BowiE and
others" compared sulfisoxazole (predominantly antichlamydial activity), with spectinomycin or
streptomycin (predominantly anti-ureaplasma activity). The results suggested that Chlamydia-negative, ureaplasma-positive urethritis responded better to spectinomycin or streptomycin, indicating a possible role for ureaplasmas in Chlamydia-negative urethritis. Should the female contact be investigated and treated? There is no evidence that contact-tracing as practised with gonorrhoea would reduce the incidence of N.G.U. There is, however, ample evidence of colonisation of female contacts with C. trachomatis,1O,l1 and most of them will be symptomless. Some salpingitis may have a chlamydial aetiology,7 whilst a third of one series of neonatal eye infections were chlamydial. These are two good reasons for treating women with proven C. trachomatis infections, and contacts of men with chlamydial urethritis.
NEONATAL HYPOGLYCÆMIA AND NESIDIOBLASTOSIS NEONATAL hypoglycaemia is usually transient and controllable, by frequent meals or by intravenous glucose. The syndrome is common, especially in small-for-gestational-age infants and in the offspring of mothers with diabetes or erythroblastosis. Persistent hypoglycaemia of infancy and childhood is much less common. It may be familial-and as such the predominant manifestation of multiple endocrine adenomatosis’—or non-familial. 25. Oriel, J. D., Ridgway, G. L., Tchomouroff, S. Scott. med. J. (in the press). 26. Handsfield, H. H., Alexander, E. R., Wang, S. P., Pedersen, A. H. B., Holmes, K. K. J. Am. vener. Dis. Ass. 1976, 2, 5. 27. Bowie, W. R., Floyd, J. F., Miller, Y., Alexander, E. R., Holmes, J., Holmes, K. K. Lancet, 1976. ii, 1276 1. Vance, J. E., Stoll, R. W., Kitabchi, A. F., Buchanan, K. D., Hollander, D., Williams, R. H. Am. J. Med. 1972, 52, 211.
mental system. It is based on the old observation that under suitable conditions the interaction of cysteine, iron, and atmospheric oxygen leads to the formation of a transient coloured complex. Mainly by virtue of its nitro group, metronidazole can take the place of oxygen in this reaction, the cysteine/ iron/drug complex decaying through free-radical intermediates. The inhibitory effect of zinc on the oxygen consumption of the cysteine/iron mixture was described in 1909 by MATHEWS and WALKER.16 More recent studies by WILLSON have shown that zinc may also inhibit the analogous metronidazoledependent free-radical sequence.4,17 Very little can be taken for granted with either trace elements or free radicals. After several decades of zinc studies in the laboratory the first human disease which can be specifically attributed to zinc deficiency came as a surprise to most, perhaps all, workers in the field.l8 Acrodermatitis enteropathica is a rare inborn metabolic disorder; and how exactly the zinc deficiency arises in the patients is still uncertain. But at a highly speculative level it is perhaps relevant that the disease affects a tissue which is most continuously exposed to ultraviolet irradiation. (In the same speculative vein one may note that of all human organs it is the eye which has the highest zinc content; and that seminal fluid, the body fluid with the highest zinc concentration, is the only one in which cells must survive outside the body if they are to fulfil their
physiological function.) Zinc may prove
be rather less prominent in protecting against iron-catalysed free-radical dammage than has been suggested, but if the suggestion catalyses a "radical" approach to pathological and medical problems it will have served a useful purpose. Such an approach might prove the more opportune in the light of recent methodological advances. Most free radicals are far too short-lived to be detectable in tissues (although free-radical signals have been recorded in cataractous lensesI9); and even the primary products of free-radical reactions tend to be unstable. For several decades almost the only chemical "marker" of biological free-radical activity has been malonyldialdehyde or M.D.A., a secondary breakdown product of polyunsaturated lipids destroyed by free-radical oxidation.2o,21 (In fact, M.D.A. is only one of many freeradical-oxidation products which give the same characteristic colour reaction with thiobarbituric acid: hence the alternative term of T.B.A.-reactive compounds.22) It has been a useful tool but it has severe limitations, especially when applied to to
16. Mathews, A. P., Walker, S. J. biol. Chem. 1909, 6, 312. 17. Willson, R. L. Lancet, 1976, i, 1407. 18. Moynahan, E. J. ibid. 1974, i, 399. 19. Weiter, J. J., Finch, E. D. Nature, 1975, 254, 536. 20. Kohn, H. I., Liversedge, M. J. Pharmacol. 1944, 82, 292. 21. Stocks, J., Dormandy, T. L. Br. J. Hœmat. 1971, 20, 95. 22. Gutteridge, J. M. C., Stocks, J., Dormandy, T. L. Anal. chim. 70, 107.
Acta, 1974,
human pathology. More recent work suggests that fluorescence spectroscopy may provide a better insight into in-vivo free-radical processes.23 The fluorescent spectra of complex biological mixtures are often difficult to interpret and to quantitate; but the difficulties may prove to be a challenge rather than a deterrent.
Treatment of
Chlamydia trachomatis
Infections Chlamydia trachomatis are common. Hyperendemic trachoma affects an estimated 500 million people in developing countries.’ In England and Wales, upwards of 70 000 cases of non-gonococcal urethritis (N.G.u.) are reported annually, of which about half will be caused by INFECTIONS with
C. trachomatis.2,3 To these must be added paratrachoma,ophthalmia neonatorum,S pneumonitis of
infants,6 salpingitis,7 "non-specific" genital infection in women,8 and post-gonococcal urethritis.9 There is now considerable evidence that the cervix of female contacts of men with N.G.U. forms a substantial reservoir of C trachomatis,10 whilst the incidence of concurrent C. trachomatis infection in women with gonococcal cervicitis may be as high as 62%." The diversity and scale of chlamydial infections has stimulated laboratory studies initially to simplify isolation of the organism, and more recently to investigate the in-vitro effects of antimicrobial agents against C. trachomatis, with a view to rationalising chemotherapy. Early in-vitro work was done with the yolk-sac of embryonated hen’s eggs.12 This technique is cumbersome and not suitable for rapid screening, either of antimicrobials or of large numbers of C. trachomatis isolates. Cell-culture provides a more convenient way of investigating the action of antimicrobials on this organism. RIDGWAY, OWEN, and ORIEL" examined the activity of eight antimicrobials on a single strain of C. trachomatis. The most active compounds were erythromycin and oxytetra23. Dillard, C. J., Tappel, A. K. Lipids, 1973, 8, 183. 1. Lancet, 1977, ii, 857. 2. Holmes, K. K., et al. New Engl. J. Med. 1975, 292, 1199. 3. Oriel, J. D., Reeve, P., Wright, J. T., Owen, J. Br. J. vener. Dis. 1976, 52, 46. 4. Darougar, S., Jones, B. R., Kinnison, J. R., Vaughan Jackson, T. D., Dunlop, E. ibid. 1972, 48, 416. 5. Rees, E., Tait, I. A., Hobson, D., Byng, R. E., Johnson, F. W. A. ibid. 1977,
53, 173. 6. Beem, M. O., Saxon, E. M. New Engl. J. Med. 1977, 296, 306. 7. Mårdh, P. A., Ripa, T., Svensson, L., Weström, L. ibid. 1977, 296, 1377. 8. Rees, E., Tait, I. A., Hobson, D., Johnson, F. W. A. in Non Gonococcal Urethritis and Related Infections (edited by D. Hobson and K. K. Holmes); p. 67. American Society for Microbiology, Washington, D.C., 1977. 9. Oriel, J. D., Reeve, P., Thomas, B. J., Nicol, C. S. J. infect. Dis. 1975, 131, 376. 10. Oriel, J. D., Powis, P. A., Reeve, P., Miller, A., Nicol, C. S. Br. J. vener. Dis. 1974, 50, 11. 11. Hilton, A. L., Richmond, S. J., Milne, J. D., Hindley, F., Clarke, S. K. R. ibid. 1974, 50, 1. 12. Werner, G. H. Ann. Inst. Pasteur, 1961, 100, 93. 13. Ridgway, G. L., Owen, J., Oriel, J. D. J. antimicrob. Chemother. 1976, 2, 71.
193 TREHARNE et al. 14 found
rifampicin more active than erythromycin or oxytetracycline, whilst both groups found sulphonamides, trimethoprim, chloramphenicol, and the penicillins only moderately active. Spectinomycin, metronidazole, and aminoglycosides were inactive. The experience of Kuo, WANG, and GRAYSTON1S was similar: they also noted that rosamicin (a new macrolide antibiotic) was just as active as erythromycin, that different
cycline.
serotypes of C. trachomatis varied in sensitivity to sulphonamides, and that penicillin gave rise to abnormal forms of C. trachomatis. Latency is a well-known phenomenon in C. psittaci infections, and has been suggested as an explanation for some of the clinical findings in oculogenital infections with C. trachomatis.16.17 In vitro, this phenomenon has been seen in C. psittaca’’8 and C. trachomatis.19 Whilst latency was particularly striking with the cell-wall inhibiting antibiotics and the sulphonamides, RIDGWAY et al. 19 found that all antimicrobials tested could induce latency after serial passage. Clearly, caution is required in the interpretation of sensitivity tests with C. trachomatis on single passage. Closely related antimicrobials may have widely differing inhibitory concentrations; thus, results with a single antimicrobial should not be taken to apply to related antimicrobials. Ocular chlamydial infections respond to 5 or 6 weeks’ treatment with tetracycline or rifampicin eye ointment. In 1972, when the role of C. trachomatis in N.G.u. was still in doubt, WILLCOX2o concluded on clinical grounds that ampicillin, nalidixic acid, and novobiocin were no better than placebo; and other compounds of questionable value included sulphonamides, penicillin, chloramphenicol, metronidazole, and spectinomycin. These findings are now borne out by in-vitro tests on C. trachomatis. Tetracyclines, by contrast, have long been known to be effective for N.G.u., and eradication of C. trachomatis coincides with resolution of symptoms.21,22 In patients with gonococcal urethritis ORIEL and his co-workers,9.23,24 showed that coinci dent infection with C. trachomatis was unaffected by gentamicin, ampicillin, and spectinomycin. These patients went on to acquire postgonococcal 14. Treharne, J. D., Day, J., Yeo, C. K., Jones, B. R., Squires, S. in Non Gonococcal Urethritis and Related Infections (edited by D. Hobson and K. K. Holmes); p. 214. American Society for Microbiology, Washington, D.C., 1977. 15. Kuo, C. C., Wang, S. P., Grayston, J. T. Antimicrob. Agents Chemother.
1977, 12, 80. 16. Hannah, L., Dawson, C. R., Briones, O.,
mun. 1968, 101, 43. 17. Richmond, S. J., Hilton,
A. L.,
Clarke,
Thygeson, P., Jawetz,
S. K. Br.
J.
vener.
Dis.
E.
J.
Im-
1972, 48,
437. 18. Hatch, T. P. Infect. Immun. 1975, 12, 211. 19. Ridgway, G. L., Owen, J., Oriel, J. D. Br. J. vener. Dis. (in the press). 20. Willcox, R. R. ibid. 1972, 48, 137. 21. Oriel, J. D., Reeve, P., Nicol, C. S. J. Am. vener. Dis. Ass. 1975, 2, 17. 22. Prentice, M. J., Taylor-Robinson, D., Csonka, G. W. Br. J. vener. Dis. 1976,
52, 269. J. D., Ridgway, G. L., Reeve, P., Beckingham, D. C., Owen, J. J. infect. Dis. 1976, 133, 568. Oriel, J. D., Ridgway, G. L., Tchomouroff, S., Owen, J. Br. J. vener. Dis.
23. Oriel, 24
1977, 53, 226.
urethritis; clearly, when treatment schedules for gonorrhoea are revised, we ought to consider a regimen effective against both Neisseria gonorrhcece and C. trachomatis. Some workers have recommended 3 weeks’ treatment for N.G.U., but it may be unrealistic to expect patients to persevere with medication every six hours for this long, when for much of the time they will be symptom-free. ORIEL et al. 25 found that 250 mg of oxytetracycline sixhourly for 2 weeks gave an 86% cure-rate, as did 500 mg of erythromycin stearate twelve-hourly for 2 weeks. Rifampicin has yet to be evaluated in the treatment of N.G.U. Whether single-dose chemotherapy is feasible (as now practised for gonococcal urethritis) remains to be seen. Treatment with antimicrobials has been employed with some success in the elucidation of other possible agents of N.G.U. HANDSFIELD et a1.26 found that the response of N.G.U. to tetracycline therapy was better in Chlamydia-positive patients than in Chlamydia-negative patients. BowiE and
others" compared sulfisoxazole (predominantly antichlamydial activity), with spectinomycin or
streptomycin (predominantly anti-ureaplasma activity). The results suggested that Chlamydia-negative, ureaplasma-positive urethritis responded better to spectinomycin or streptomycin, indicating a possible role for ureaplasmas in Chlamydia-negative urethritis. Should the female contact be investigated and treated? There is no evidence that contact-tracing as practised with gonorrhoea would reduce the incidence of N.G.U. There is, however, ample evidence of colonisation of female contacts with C. trachomatis,1O,l1 and most of them will be symptomless. Some salpingitis may have a chlamydial aetiology,7 whilst a third of one series of neonatal eye infections were chlamydial. These are two good reasons for treating women with proven C. trachomatis infections, and contacts of men with chlamydial urethritis.
NEONATAL HYPOGLYCÆMIA AND NESIDIOBLASTOSIS NEONATAL hypoglycaemia is usually transient and controllable, by frequent meals or by intravenous glucose. The syndrome is common, especially in small-for-gestational-age infants and in the offspring of mothers with diabetes or erythroblastosis. Persistent hypoglycaemia of infancy and childhood is much less common. It may be familial-and as such the predominant manifestation of multiple endocrine adenomatosis’—or non-familial. 25. Oriel, J. D., Ridgway, G. L., Tchomouroff, S. Scott. med. J. (in the press). 26. Handsfield, H. H., Alexander, E. R., Wang, S. P., Pedersen, A. H. B., Holmes, K. K. J. Am. vener. Dis. Ass. 1976, 2, 5. 27. Bowie, W. R., Floyd, J. F., Miller, Y., Alexander, E. R., Holmes, J., Holmes, K. K. Lancet, 1976. ii, 1276 1. Vance, J. E., Stoll, R. W., Kitabchi, A. F., Buchanan, K. D., Hollander, D., Williams, R. H. Am. J. Med. 1972, 52, 211.
