443

hibitor, L-deprenil, will potentiate the anti-akinetic

A NEW TYPE OF PENICILLIN RESISTANCE OF

properties of levodopa, and that it is the availability of D.A. which is responsible for the therapeutic effect.

STAPHYLOCOCCUS AUREUS*

We thank the Chinon Drug Company and Prof. pest for the supply of L-deprenil.

J. Knoll of Buda-

Requests for reprints should be addressed to M. B. H. Y., Israel Institute of Technology-Technion, School of Medicine, Department of Pharmacology, 12 Haaliya Street, Bat-Galim, P.O.B. 9649, Haifa, Israel.

L. D. SABATH NANCY WHEELER MICHEL LAVERDIERE DONNA BLAZEVIC BRIAN J. WILKINSON

Departments of Medicine, Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, U.S.A. Penicillin - "tolerant" Staphylococcus strains are resistant to the lethal action of penicillins, but are inhibited by normal (low) concentrations. They are deficient in autolytic enzyme activity which appears to be necessary for bacteriolysis and the lethal action of penicillins. This "deficiency" is caused by a large excess of an inhibitor of autolysin. Seven such tolerant strains have been isolated from blood, bone, or sputum of patients who responded poorly to penicillin treatment of endocarditis, osteomyelitis, or staphylococcal pneumonia. These isolates were of different phage-types, and most showed cross-tolerance to the killing action of cephalosporins or vancomycin, antibiotics to which they were sensitive (inhibited). They were killed at normal rates by gentamicin, cycloserine, and rifampicin. Population analysis indicated that the proportion of tolerant organisms within a resistant strain is 7% or less; their ability to inhibit autolytic activity within their own and neighbouring cells appears to account for the net decreased autolytic activity of the entire strain. 44% of the bacteræmic strains studied showed penicillin tolerance. Tolerance is thus a common, clinically important form of penicillin resistance, that differs from previously described forms of penicillin resistance, that due to &bgr;-lactamase, and that due to "intrinsic" (e.g., methicillin resistance) mechanisms.

Summary

REFERENCES 1. Birkmayer, W., Mentasti, M. Arch. Psychiat. Z. ges. Neurol, 1967, 210, 29. 2. Birkmayer, W., Neumayer, E. Z. Neurol. 1972, 202, 257. 3. Cotzias, G. C.J. Am. med. Ass. 1971, 218, 1903. 4. Barbeau, A.Adv. Neurol. 1973, 2, 173. 5. Birkmayer, W., Hornykiewicz, O. Unpublished. 6. Birkmayer, W., Riederer, P., Linauer, Youdim, M. B. H. J. neural Transm.

1975, 36, 303. 7. Ludin, H.P., Bass-Verrey, R. ibid. 1976, 38, 249. 8. Diamon, G., Markham, C. ibid. p. 259. 9. Lancaster J., Sawyer, P. R., Shepherd, D. M., Turnbull, M.

J. Br. J. Pharmac. 1973, 47, 838. 10. Wurtman, R. J., Romero, J. A. Neurology, Minneap. 1972, 22, suppl., 72. 11. Zeller, E. A., Boshes, B., Arbit. J., Bieber, M., Blonsky, E. R., Dolkart, M., Huprikar, S. V.J. neural Transm. 1976, 39, 63. 12. Youdim, M. B. H. in Monoamine Oxidase and Its Inhibition (edited by G. E. W. Wolstenholme and J. Knight); p. 405. Amsterdam, 1976. 13. Youdim, M. B. H. in Research Methods in Neurochemistry (edited by N. Marks and R. Rodnight); p.167. New York, 1975. 14. Lowry, 0. H., Rosebrough, N.J., Farr, A. L., Randall, R. J. J. biol. Chem. 1951, 193, 265. 15. Riederer, P., Wuketich, H. J. neural Transm. 1976, 38, 277. 16. Birkmayer, W., Riederer, P., Youdim, M. B. H. Unpublished. 17. Murphy, D. L., Donnelly, C. H. Adv. Biochem. Psychopharmac. 1974, 12, 71. 18. Youdim, M. B. H., Grahame-Smith, D. G., Woods, H. F. Clin. Sci. molec.

Med. 1976, 50, 479. 19. Murphy, D. L., Wyatt, R. J. Nature, 1972, 238, 225. 20. Youdim, M. B. H., Holzbauer, M. J. neural Transm. 1976, 38, 193. 21. Youdim, M. B. H. in Neuroregulators and Hypothesis of Psychiatric Disorders (edited by E. Usdin, D. Hamberg, and J. Barchas); Oxford, 1977 (in the press). 22. Glover, V., Sandler, M., Owen, F., Riley, G. J. Nature (in the press). 23. Ehnnger, H., Hornykiewicz, O. Klin. Wschr. 1960, 38, 1236. 24. Birkmayer, W., Hornykiewicz, O. ibid. 1961, 73, 787. 25 Barbeau, A. Archs Neurol. 1961, 4, 97. 26. Birkmayer, W., Hornykiewicz, O.Archs Psychol. 1962, 203, 506. 27. Houslay, M. D., Tipton, K. F., Youdim, M. B. H. Life Sci. 1976, 19, 467. 28. Youdim, M. B. H., Birkmayer, W., Riederer, P. in Problems in Extrapyramidal Disorders (edited by M. Trabucchi and P. Spano) New York, 1977 (in the press). 29. Knoll, J. and Magyar, K. Adv. biochem. Psychopharmac. 1972, 5, 393. 30. Knoll, J. in Monoamine Oxidase and Its Inhibition (edited by G. E. W. Wolstenholme and J. Knight); p. 135. Amsterdam, 1976. 31. Long, R. Personal communications. 32. Birkmayer, W., Riederer, P. J. neural Transm. 1975, 37, 175. 33. Christmas, A.-J., Coulson, C. J., Maxwell, D. R. and Riddell, D. Br. J. Phar-

mac. 1972, 45, 490. 34. Green, A. R., Youdim, M. B. H. ibid. 1975, 55, 415. 35. Green, A. R., Mitchell, B., Tordoff, A., Youdim, M. B. H. ibid. (in the press). 36. Knoll, J., Ecseri, Z., Kelemeu, K., Nievel, J. and Knoll, B. Archs int. Pharmacodyn. Thér. 1965, 155, 154. 37. Fuxe, K. and Ugerstedt, U. in Amphetamine and Related Compounds (edited by E. Costa and S. Garrattini); p. 257. New York, 1970. 38. Yang, H-Y. T., Neff, N. H. J. Pharmac. exp. Ther. 1974, 189, 733. 39. Fischer, E., Spatz, A., Heller, B., Reggiani, H. Experientia, 1972, 28, 307. 40. Boulton, A. A., Juorio, A. V., Phillips, S. R. Wu, P. H. Brain Res. 1975, 96, 212. 41. Boulton, A. A., Baker, G. P. J. Neurochem. 1975, 25, 477. 42. Inwang, E. E., Mosnain, A. D., Sabelli, H. C. ibid. 1973, 20, 1469. 43 Levine, R. J., Nirenberg, P. Z., Udenfriend, S., Sjoerdsma, A. Life Sci. 1964,

3, 651. 44. Neff, N. H. and Fuentes, A. in Monoamine Oxidase and its Inhibition (edited by G.E. W. Wolstenholme and J. Knight); p. 163. Amsterdam, 1976. 45. Mantegazza, P., Riva, M.J. Pharmac. 1963, 15, 472. 46. Nakajima, T., Kakimoto, Y., Sano, J. J. Pharmac. exp. Ther. 1964, 143, 319. 47 Dzolzic, M.-R., Brunivels, J., Bonta, J. L. J. neural Transm. (in the press). 48 Braestrup, C., Andersen, H., Randrup, A. Eur. J. Pharmac. 1975, 34, 181. 49. Baker, G. B., Raiteri, M., Bertollini, A., Del Carmine, R., Keane, P. E., Martin, I. L. J. Pharm. Pharmac. 1976, 28, 456. 50. Horn, A. G. Br. J. Pharmac. 1973, 47, 332. 51 Hornykiewicz, O. Br. med. Bull. 1973, 29, 172. 52. Sandler, M., Bonham-Carter, S., Hunter, K. R., Stern, G. M. Nature, 1973,

241, 439. 53. Sourkes, T.L. Biochem. Med. 1970, 3, 321.

aureus

Introduction THE first form of bacterial resistance to penicillins identified was that caused by penicillinase (&bgr;-lactamase, E.C.3.5.2.6).’ Penicillinases have been identified in almost all bacteria (including the tubercle bacillus)l that are highly resistant to benzylpenicillin and this is probably the most common mechanism of resistance to penicillins and cephalosporins. A second form of resistance to penicillins is "intrinsic" resistance, that not caused by drug inactivation.3It was first noted in the laboratory in resistance induced by multiple transfers in the presence of low concentrations of penicillin,4but its clinical im11 portance was first noted in gram-negative bacillis when it became apparent that &bgr;-lactamase alone could not account for the resistance observed. Perhaps the purest form of intrinsic resistance to p-lactam antibiotics is that of penicillinase-negative segregants of methicillin-resistant S. aureus ; these organisms are highly resistant to methicillin (and other penicillins and cephalosporins), but produce no -lactamase at all .7 8 Penicillin tolerance, a third form of penicillin resistance, differs from the other two mechanisms of resistance in that the minimum inhibitory concentration

(M.!.c.) of penicillins for the tolerant organisms is low (sensitive range), but the minimum bactericidal concen*Presented in part at the 16th Interscience Conference bial Agents and Chemotherapy, Chicago, Oct. 28, 1976.

on

Antimicro-

444 TABLE I—PHAGE TYPES OF TOLERANT S. AUREUS STRAINS

with chronic

staphylococcal pneumonia. The seven strains epidemiologically distinct and had different phage types (table i). An eighth penicillin-tolerant organism, originally 180lated from a patient in Georgia 13 was obtained from Dr George Warren, Philadelphia. The control strain was the were

"Oxford" S.

aureus.

Antibiotics Each of the antibiotics was donated by its manufacturer: methicillin by Bristol Laboratories, Syracuse; nafcillin by

Wyeth Laboratories, Philadelphia; cephalothin, vancomycin, and cycloserine by Eli Lilly Laboratory for Clinical Research, Indianapolis; clindamycin by the Upjohn Co., Kalamazoo; gentamicin by the Schering Corporation, Bloomfield, New Jersey ; and rifampin by Ciba-Geigy, Summit, New Jersey.

R.T.D.=Routine test dilution.

*Carrying own lysogenic phage.

Susceptibility Tests The

tration

(M.B.c.) is high, often more than 100 times the MJ.c. Although one of the many desirable attributes of penicillin is that it kills bacteria at almost the same concentration at which they are inhibited," the recognition of tolerance provides one exception. Although the term "penicillin-tolerant" was used by BarberIO in describing methicillin-resistant staphylococci (described above as the second form), the term "tolerant" is used here because the phenomenon observed is so much like the "tolerance" in pneumococci, described by Tomasz et al. 11 The tolerant organisms described here are not the same as the "persisters" described by Bigger12 for when are killed after 24 h the persisters are retested >99-9% exposure to penicillins. Materials and Methods

Organisms The 7 tolerant organisms studied in detail were isolated from bone of two patients (a 19-year-old male with recurrent osteomyelitis of the femur and from a 62-year-old woman with 8 years of osteomyelitis at various sites) and from the blood of four patients: a 13-month-old infant following surgery for transposition of the great vessels, a 16-month-old infant with an extensive 3rd-degree burn, a third child (5 years old) with endocarditis, and a 39-year-old man with hepatitis-B infection, progressive liver failure, and bacteraemia from an unknown source. A seventh was isolated from the sputum of a patient

*Methicillin-resistant strain, tolerant

to

determined by adding approximately 5×105 units colony-forming (c.F.u.) in 0.5 ml from a 4 h culture (colony counts were done on each inoculum) to each of a series of tubes containing 2-fold dilutions of the antibiotic to be tested in Mueller-Hinton broth (Difco), and incubating for about 24 h at 37°C. Visual inspection revealed the tube with the lowest concentration of antibiotic (M.iC.) showing no growth (no turbidity). After 24 h incubation, and also after 48 h incubation, 0.1 ml was removed from each clear tube (after thorough mixing) and spread on the surface of ’Trypticase’ soy agar (T.S.A.) plates, and incubated at 37°C for 24-48 h, to determine the surviving c.F.u. The percent killed could thus be calculated; 50 or fewer c.F.u. per 0.1ml subcultured represented 999% or greater reduction in c.F.u., the definition of "killing" used here, as suggested by the American Society for m.i.c. was

Microbiology. 14 Autolysin Activity Autolysin for determining the level of autolytic activity in the various strains of S. aureus was prepared as described by Best et al.;13 the crude autolysin was obtained from cells by

freeze-thaw treatment. The substrates used here were whole cells of S. aureus ATCC 6538P that had been grown in a broth of 5 g ’Bacto-peptone’ (Difco), 5 g yeast extract (Difco), 2g glucose, and 1 g dipotassium phosphate per litre of distilled water to which 0.05 p.Ci/mf[3H]-glycine had been added. After growing for 18 h at 37°C, the cells were centrifuged at 1650 g, washed three times with distilled water, resuspended in 10% trichloracetic acid (T.C.A.), and heated at 95C for 20 min. The cells were washed five times with distilled water, resuspended

cephalothin and vancomycin (see table III).

445 at a concentration and 000-100 000 80 kept at -20°C in c.p.m./ml, yielding small aliquots (5 ml) until needed for assay. The assay was for liberation of the 3H-marker from the insoluble substrate on addition of autolysin; 13 negligible release occurred in control tests to which no autolysin had been added. An additional substrate tested was chemically prepared peptidoglycan, labelled as noted above, but from which non-peptidoglycan material was removed by extraction with cold T.C.A., aqueous ethanol, hot T.C.A., and trypsin treatment. 13 Aminoacid analysis of the

in 0.01

moVI phosphate buffer pH 7.0

TABLE IV—CHANGE IN AUTOLYTIC ACTIVITY OF S. AUREUS AFTER TO GROWING CULTURES ADDITION OF NAFCILLIN

(1.6 µg/ml)

peptidoglycan showed glutamic acid, lysine, alanine, glycine, serine, muramic acid, and glucosamine present in molar ratios of 1.0, 0.83, 1.5, 4.25, 0.21, 0.39, and 0.55, respectively. The fractional values for glucosamine and muramic acid were caused by instability during the acid hydrolysis before aminoacid analysis. Lysis of Growing Bacteria Rates of lysis of staphylococci on addition of antibiotic were determined using 18 h cultures in Mueller-Hinton broth grown

*Each number expresses results of a different experiment. Activity was measured as c.p.m. per µg protein released from 3H-labelled wholecell, or peptidoglycan substrate, but autolysin preparation. See Methods for details.

tNafcillin-resistant-25 µg/ml nafcillin used. N.T.=not

TABLE III-STAPHYLOCOCCAL CROSS-TOLERANCE TO CEPHALOTHIN

tested.

AND/OR VANCOMYCIN

.

*24 h incubation. tData from table n.

*Melhicillin-resistant strain. No cross-tolerance was noted with cycloserine, gentamicin, pin. Variable results were noted with clindamycin (data

or

rifam-

not

longed (48 h) incubation resulted in additional "killing" (a reduction in c.F.u. of -99-9% or more) with some strains (table n). None was tolerant to the killing action of cycloserine, gentamicin, or rifampin. Table iv lists autolytic activity changes of six tolerant strains comparing rates just before nafcillin (-1 6µg/ml) was added to rapidly growing cultures and maximum activity over 120 min afterwards. Thus, as high as a 9.4-fold increase in autolytic activity was noted when nafcillin was added to the control strain, but the penicillin-tolerant strains tested showed a minimum (15% at most) or no increase in autolytic activity. All strains produced some autolytic activity, however, causing ten or more times as much radioactivity to be released from the insoluble cell substrate as occurred spontaneously in the absence of autolysin.

pre-

sented). at 370 in a rotary shaker, and adding 1% vol/vol to additional broth as inoculum. After rapid growth (o.D.=0-4) was noted (Bausch and Lomb Spectrophotometer 20 at 620 nm) antibiotic was added and subsequent optical density readings were taken at appropriate intervals.

Results The M.t.c. and M.B.c. test results are summarised in tables II and ill which show six of the seven patientstrains had M.LC. values indicating that they were susceptible to the semisynthetic penicillin nafcillin, and all were susceptible to cephalothin as were the Evans and control strains, with M.LC. values of 0.to 0 4 µg/ml for nafcillin. All these strains (except the third) were susceptible to methicillin. However, the M.B.C. values for the penicillin-tolerant strains were 128 to 2000-fold higher, whereas the control strain’s M.B.C. was only four times the M.t.c. However, the 48 h M.B.C. test (table II) revealed bactericidal activity at or near the M.i.c. values for all but four strains (strains 2, 4, 6, and 7). Table ill shows that all the strains tolerant to the killing action of nafcillin were also tolerant, compared to the control strain, to the same action of vancomycin or cephalothin in a 24 h bactericidal test, but that pro-

Fig. 1-Autolytic activity of 2 tolerant strains and control (Oxford) strain of S. aureus after addition of nafcillin (16

µg/ml). Specific activity is counts per minute of ’H label released from ’H-labelled peptidoglycan (prepared from S. aureus, Oxford) per minute per ug protein obtained from cells after addition of nafcillin at time zero, O-O=S. aureus Oxford (control); 0 ... O=S. aureus 4; 0 - - - O=S. aureus Evans. The 2 tolerant strains did not show an increase whereas control strain showed 6-fold increase in autolytic activity.

446 1 compares autolytic activity of the control strain of the penicillin-tolerant strains before, and over 120 min after the addition of nafcillin and fig. 2 shows the rates of lysis of whole cultures of tolerant and susceptible strains. There was a delay in the appearance of autolytic activity (fig. 1) and also a delay (of several hours) before lysis of whole cells occurred after nafcillin was added to the control strain and tolerant strains. The

Fig.

and

two

rate of growth of the two tolerant strains was slightly faster than that of the control strains before the addition of nafcillin. Fig. 3 shows that equal amounts of some "autolysin" preparations (obtained either from tolerant organisms after addition of nafcillin, or control cells in the absence of antibiotic) added to the activated (by addition of nafcillin) control autolysin material inhibited autolysin activity. The inhibitory effect reduced total activity to less than one-tenth of that seen without the inhibitory substance. Such inhibitory preparations could be diluted 1/1000 and still produce the same degree of inhibition. The inhibitory substance was heat-labile. Retrospective review of M.LC. and M.B.c. values for bacteraemic isolates of S. aureus in the diagnostic microbiology laboratory at University of Minnesota Hospitals indicated that 44% of the isolates (28 of 63) tested in the last year showed tolerance (M.B.C./M.I.C. >32).

Discussion The identification of penicillin-tolerant staphylococci from these patients with staphylococcal bacteraemia,

Fig. 3-Inhibition of cell-wall autolytic activity of S. aureus. Left panel shows inhibition of autolytic activity by addition of material from tolerant strain S. aureus Evans. (Specific activity as defined in fig. 1). The autolytic activity of control strain S. aureus Oxford (0 - - - 0) increased about 9-fold 90 min after addition of nafcillin, 1.6g/ml. S. aureus Evans (0 ... 0) showed no increase m autolytic activity over 90 min following addition of 1.6 µg/ml, and mixture of equal volumes of each (0-0) completely inhibited increase in autolytic activity of control preparations. Right panel shows that autolytic activity of control strain S. aureus Oxford (0 - - - 0) produced by addition of nafcillin, 1.6µg/ml, to logphase strain increased 9-fold whereas the same strain without nafcillin added (0-0) showed no increase in autolytic activity during comparable period (slight decrease was observed). Mixture of equal volumes 0 ... 0 completely obliterated increase in autolytic activity produced by nafcillin. Theoretical activity expected at 90 min if activities were additive would have been 200 c.p.m./µg protein (i.e., 360+40-2).

osteomyelitis, ment

or

had been

a

pneumonia, in whom antibiotic treatproblem, indicates that the slow rates

of killing might well have been a critical factor in each clinical course. Defects in rates of killing have been identified elsewhere,16 17 which indicates that the problem is widespread. Tests of bactericidal rate might help to indicate when additional drugs (e.g., those that act by different mechanisms) should be added, therapy be radically changed, or the duration of therapy be substantially prolonged (measures taken in these patients). Dissociation of inhibitory (M.I.c.) and bactericidal (M.B.C.) functions-even though substantial (128 to 2000-fold differences)-disappeared on prolonged incubation (48 h) in some strains. This indicates that the bactericidal action or a killing process operates in the tolerant strains, but at a slower rate, not caused by a slower rate of growth of tolerant strains, for they grew as rapidly as the control strain (or possibly more rapidly,

[fig. 3]).

Fig. 2-Lysis of control and tolerant S. aureus by nafcillin. Lysis (change in O.D. at 620 nm) of control (Oxford) strain and 2 tolerant strains (S. aureus 4 and S. aureus Evans) after addition of nafcillin (16 µg/ml) to rapidly growing cells in Mueller-Hinton broth, at time indicated by arrows. []—[]=S. aureus Oxford; •. . . •=S. aureus 4; 0 - - - O=S. aureus Evans. Control strain lysed more rapidly and tolerant strain grew slightly faster during logarithimic growth-phase.

The occurrence ofpenicillin tolerance in S. aureus strains causing serious disease in patients probably represents a naturally occurring counterpart to the penicillin tolerance of pneumococci (i.e., inhibition, but not bacterial killing) created in the laboratory by Tomasz et al. who produced tolerance, either by growth of pneumococci in special medium, which led to abnormal teichoic acids," or by the use of chemical mutagens to produce an autolysin-deficient mutant.18 Their tolerant pneumococci showed cross-tolerance to many other cellwall-active antibiotics-i.e., vancomycin, cycloserine, bacitracin, phosphonomycin-possibly because of a sin-

447 TABLE V-A COMPARISON OF

3

EARLY MORNING MIGRAINE Nocturnal Plasma Levels of Catecholamines, Tryptophan, Glucose, and Free Fatty Acids and

TYPES OF PENICILLIN RESISTANCE

OF S. AUREUS

Sleep Encephalographs L. K. G. HSU R. S. KALUCY

A. H. CRISP J. KOVAL C. N. CHEN

St.

George’s Hospital Medical School M. CARRUTHERS

St.

Mary’s Hospital Medical School

K. J. ZILKHA National Hospital for Nervous Diseases, London

’On storage of

(tolerance), loss of S.

4C there is gradual loss of resistance This may represent a plasmid locus (c.f. &bgr;-lactamase plasmid on such storage,26 in some

organisms

over

4-12

aureus

at

mo.

strains). tAt least 5 phage-types, 3

groups

(see table I).

in pneumococci 18 whereas that reported frequent cross-tolerance to vancomycin or cephalothin, but usually not to both (table in), possibly due to 3 different cell-wall autolysins-i.e., an N-acetylglucidase, an amidase, and an endopeptidase

gle autolysin here showed

.

-in S. aureus. 19 These results support the suggestions of Weidel and Pelzer, Tomasz, Rogers, and their co-workers" 2U-2.:1 that autolysins play a vital role in the lethal action of penicillin on bacteria. The. fact that achievement of 99.9% killing in 24 h is associated with a six to nine fold increase in autolytic activity, as shown here and by Best et al.," indicates that the amount of autolytic activity present in rapidly growing S. aureus is not adequate to cause rapid killing of S. aureus. Inhibitor(s) of autoly-

sins, (possibly lipoteichoic acid) 24

25

are obviously important ; their identity and control require further inves-

tigation. A comparison

of the characteristics of tolerance with those of the two previously recognised forms of penicillin resistance of S. aureus (&bgr;-lactamase mediated, and intrinsic resistance) is summarised in table v. Further elucidation of this phenomenon may help add to our knowledge of how some antibiotics work, and in what diseases bactericidal activity is desirable. We thank Ken Dorian for phage Abraham for technical assistance.

typing

the

organisms,

and Leah

Requests for reprints should be addressed to L. D. S., Department of Medicine, University of Minnesota, Mayo Memorial Building, 420 Delaware Street S.E., Minneapolis, Minnesota 55455, U.S.A. REFERENCES 1 Abraham, E. P., Chain, E. B. Nature, 1940, 146, 837. 2. Kasik,J E., Peacham, L. Biochem. Med. 1968, 107, 675. 3 Sabath, L. D., Wallace, S. J. Ann. N. Y. Acad. Sci. 1971, 182, 258. 4. Abraham, E. P., Chain, E., Fletcher, C. M., Gardner, A. D., Heatley, N. G., Jennings, M. A., Florey, H. W. Lancet, 1941, n, 177. 5. Sutherland, R. J. gen. Microbiol. 1964, 34, 85. 6 Sabath, L D., Finland, M. Ann. N. Y. Acad. Sci. 1967, 145, 237. 7 Seligman, S. J. Nature, 1966, 209, 994. 8. Dyke, K. G., Jevons, M. P., Parker, M. T. Lancet, 1966, i, 835.

Nocturnal

plasma catecholamine, tryptophan, glucose, and free fatty acid levels have been measured in 19 subjects who regularly awoke from sleep with migraine. All-night polygraphic sleep recordings were also made. 13 subjects were studied on a second occasion allowing within-subject control as well as group comparison. Plasma total catecholamine and specifically plasma-noradrenaline levels were significantly higher in the three hours before the subjects awoke with migraine. No differences were found in plasma tryptophan, glucose, insulin, and free fatty acid levels in the migraine/no-migraine categories. Waking with migraine occurred significantly more often from rapid-eye-movement (REM) sleep. There were no other differences in sleep pattern in the migraine/nomigraine categories.

Summary

Introduction

CATECHOLAMINES,1 Serotonlri,2glucose,and free fatty acids4 have been suggested as mediators in the pathogenesis of migraine. However, the activity of these substances in plasma before the onset of a spontaneous migraine have not been extensively studied. This is partly because of the lack of certainty with which one can predict the time of onset of a migraine attack. Most investigations have therefore been carried out either

9. Fleming, A. Br. J. exp. Path. 1929, 10, 226. 10. Barber, M. in Ciba Foundation Study Group No. 13, Resistance of Bacteria to the Penicillins, (edited by A. V. S. de Reuck and M. P. Cameron); p. 89. Boston, 1962. 11. Tomasz, A., Albino, A., Zanati, E. Nature, 1970, 227, 138. 12. Bigger, J. W. Lancet, 1944, ii, 497. 13. Best, G. K., Best, N. H., Koval, A. V. Antimicrob. Agents Chemother. 1974, 14.

6, 825. Barry, A. L., Sabath,

L. D. in Manual of Clinical Microbiology, (edited by E. H. Lennette, E. H. Spalding, and J. P. Truant); p. 431, American Society for Microbiology, Washington, D.C., 1974. 15. Park, J. T., Hancock, R. J. gen. Microbiol. 1960, 22, 249. 16. Gopal, V., Bisno, A. L., Silverblatt, F. J. J. Am. med. Ass. 1976, 236, 1604. 17. Mayhall, C. G., Medoff, G., Marr, J. J. Antimicrob. Ag. Chemother. 1976,

10, 707. 18. Tomasz, A., Westphal, M. Proc. natn. Acad. Sci. 1971, 68, 2627. 19. Tipper, D. J. J. Bact. 1969, 97, 837. 20. Weidel, W., Pelzer, H. Adv. Enzymol. 1964, 26, 193. 21. Tomasz, A., Waks, S. Proc. natn. Acad. Sci. 1975, 72, 4162. 22. Rogers, H. J. Nature, 1967, 213, 31. 23. Rogers, H. J., Forsberg, C. W. J. Bact. 1971, 108, 1235. 24. Holtje, Tomasz, A. Proc. natn. Acad. Sci. 1975, 72, 1690. 25. Cleveland, R. F., Wicken, A. J., Daneo-Moore, L., Shockman, G. 1976, 126, 192. 26. Barber, M. J. gen. Microbiol. 1949, 2, 274.

D. J. Bact.