CLIN. CHEM.

25/3, 484-485 (1979)

ImprovedLysozymeAssay in BiologicalFluids

N. Grossowicz,1 Mira Ariel,1 and T. Weber2

We describe a simple, rapid, sensitive, and highly repro- Materials and Methods
ducible assay for lysozyme, with use of concentrated cell
M. lysodeikticus cells were grown for 18 to 20 h in 250-mL
suspensions of Micrococcus Iysodeikticus in Tris-buffered
Erlenmeyer flasks containing 100 mL of Difco Brain Heart
glycerol/water (40/60 by vol), pH 7.5. Stored at -20 #{176}C,
Infusion Broth, with shaking, in a water-bath at 37 #{176}C.The
the cells’ susceptibility to lysozyme remains unaltered over
cells were harvested by centrifugation at 7500 X g at 4#{176}C
for
long periods. Almost identical concentration curves were 5 mm; washed with 60 mmol/L tris(hydroxymethyl)methyl-
obtained with different aliquots of the same preparation amine (Tris) buffer, pH 7.5; resuspended in the same buffer
during eight months. Lysozyme activity was reflected in containing glycerol (400 mL/L); and stored at -20 #{176}C
in small
the decrease in absorbance of the reaction mixture after plastic tubes containing suitable aliquots of the suspension.
incubation for 15 mm at 37 #{176}C. Concentrations of egg- Each tube of suspension was used only once.
white lysozyme as low as 0.02 mg/L can be accurately The lysozyme assay wasdone as follows. The cell suspension
assayed. was diluted 100-fold with the Tris buffer to give an absorbance
reading of about 200 arbitrary (Klett) units (Klett-Summer-
Additional Keyphrases: diagnosis of leukemia renal
#{149} dis- son photoelectric colorimeter with filter no. 54 and adapter
ease bacterial meningitis for 20 X 150 mm optically matched test tubes). The reaction
mixture contained 7 mL of the diluted cell suspension and
Lysozyme (EC 3.2.1.17; muramidase), a cationic enzyme of either 0.1 mL of increasing concentrations of lysozyme
low relative molecular mass (Mr = 14 500), was first described (0.005-10 mg of egg-white lysozyme, crystallized three times,
in 1922 by Fleming, who had also shown its wide distribution Grade I, Sigma Chemical Co., St. Louis, MO 63178, per liter
in tissues and body secretions (1,2). The enzyme is normally of NaCl, 8.5 g/L) or the sample to be tested (0.02-0.10 mL) in
present in plasma (5 to 9 mg/L) and only in trace amounts in a volume of 0.1 mL. The reaction mixture was incubated at
urine (3). High concentrations of lysozyme are found in leu- 37#{176}C
in a water-bath and the absorbance was determined at
kocytes, neutrophilic granulocytes and monocytes, or mac- zero time and at convenient time intervals for 30 mm.
rophages, and little if any is in lymphocytes (4).
Recently there has been increasing interest in lysozyme Results
activity as an aid in the differential diagnosis of leukemias (5,
Cells suspended in the Tris buffer containing glycerol and
6), as a diagnostic and prognostic tool in renal diseases (7), and
stored at -20 #{176}Cremained unfrozen and maintainedtheir full
in distinguishing between bacterial and viral meningitis
susceptibility toward lysozyme on repeated testing during
(8).
eight months (Figure 1). Three additional batches of cells,
In the presence of immunoglobulins and complement, ly- similarly prepared and stored for two to four months, gave
sozyme is effective against a broad variety of bacteria, in-
essentially identical results. After incubation at 37 #{176}Cfor 15
cluding some pathogens (9). The enzyme catalyzes hydrolysis mm, a decrease in absorbance of about 75% occurredata ly-
of N-acetylmuramic acid (1 -. 4) N-acetylglucosamine link-
sozyme concentration of 0.5 mg/L; decreases were about 65
ages of the polymeric chains of the bacterial cell wall. Under
and 40% at concentrations of 0.25 and 0.1 mg/L, respectively.
proper conditions this activity leads to the disruption of the
Thus the range of assayable lysozyme concentrations is from
cells, the dispersion of their contents, and the clearing of the 0.02 to 0.25 mg!L.
turbid bacterial suspension. Since the first procedure devel-
Figure 2 shows the lysis of M. lysodeikticus cell suspensions
oped by Boasson (10), most assays for lysozyme activity have
as a function of time in the presence of several concentrations
been based on the clearing phenomenon after lysis of the
of lysozyme. The absorbance decreased most within the first
sensitive microorganism, Micrococcus lysodeikticus (reviewed
10 mm of incubation, after which only residual clearing of the
in 11). In this paper we describe a simple, sensitive, and highly
cell suspension took place. Hence, a 15-mm incubation was
reproducible assay of lysozyme activity in which a concen-
used in these studies. The maximum deviation in lysis in the
trated bacterial suspension of M. lysodeikticus in glycerol!
presence of a given concentration of lysozyme in eight dif-
water (40/60 by vol) is used, which can be stored for long pe-
ferent runs did not exceed 3%.
riods of time at -20 #{176}C.
Lysozyme activity in sera from healthy subjects and some
leukemic patients, and in serum or cerebrospinal fluid from
1 Department of Bacteriology, The Hebrew University-Hadassah some patients with other diseasesis shown in Table 1.
Medical School, Jerusalem, Israel. Reprint requests should be ad-
dressed to Dr. Grossowicz. Discussion
2 The Minerva Institute for Medical Research, Helsinki, Fin-
land. Various investigators have used different methods for
Received Oct. 25, 1978; accepted Dec. 11, 1978. storingM. Iysodeikticus cells forlysozymeassay,usuallyre-

484 CLINICAL CHEMISTRY, Vol. 25, No. 3, 1979

 Table 1. Lysozyme Activity in Serum or
Cerebrospinal Fluid of Normal Subjects and
Patients with Leukemia or Meningitis
No.
Diagnosis subJects Lysozyme, mg/La
Normal, serum 10 3.4-7.2 (5.7)
LaJ Chronic myelocytic leukemia, 5 24.5-108 (68.5)
0
z serum
4 IC
Acute myelocytic leukemia, 5 17.5-31 (23.2)
a:
0 serum
(I)
Acute lymphocytic leukemia, 2 11.5; 14
4 serum
Viral meningitis, CSF 8 0.17-1.12(0.63)
Bacterial meningitis, CSF 1 42.3
a Average value in parentheses.

Suspensions of freshly harvested M. lysodeikticus cells in

LYSOZYME (mg/L) 60 mmol/L Tris buffer, pH 7.5, to which glycerol (400 mLIL)
FIg. 1. Susceptibility to lysozyme of M. lysodeiktlcus suspensions has been added, are easily prepared and maintain essentially
stored at -20 #{176}C
in glycerol/water (40/60 by vol) unaltered susceptibility to lysozyme during long storage (at
0, suspension before storage; #{149}
and , suspensions after three and eight least eight months) at -20 #{176}C. Variation between different
months of storage, respectively batches or cells or aliquots of the same batch over considerable
time intervals did not exceed 3%.

We thank Mrs. Hagit Lavi for able technical assistance. N.G. is an

established investigator of the Chief Scientist’s Office, Ministry of#{149}
Health, Government of Israel, and he is grateful to the Sigrid Jus#{233}lius
Foundation, Helsinki, Finland, for the personal grant made available
to him.

References
U 1. Fleming, A., On a remarkable bacteriotytic element found in tissues
0 and secretions. Proc.R. Soc.London, Ser. B 93, 306 (1922).
z
4 2. Imoto, T., Johnson, C. N., North, A. C. T., et al., Vertebrate lyso-
a: II zymes. In The Enzymes, 3rd ed., P. D. Boyer, Ed., Academic Press,
0 Inc., New York, NY, 1972, p 666.
U)
3. Harrison, J. F., Parker, R. W., and De Silva, K. L., Lysozymuria
4 and acute disorders of renal function. J. Clin. Pathol.26, 278
(1973).
4. Finch, S. C., Lamphere, J. P., and Jablon, S., The relationship of
serum lysozyme to leukocytes and other constitutional factors. Yale
J. Biol. Med. 36, 350 (1964).
5. Perillie, P. E., Kaplan, S. S., Lefkowitz, E., et al., Studies of mu-
ramidase (lysozyme) in leukemia. J. Am. Med. Assoc. 203, 317
(1968).
6. Osserman,E. F., and Lawlor, D. P., Serum and urinary lysozyme
10 20 30
(muramidase) in monocytic and monomyelocytic leukemia. J. Exp.
MINUTES Med. 124,921 (1966).
Fig. 2. Effect of duration of incubation at 37 #{176}C
on lysozyme- 7. Prockop, D.J., and Davidson, W. D., A study of urinary and serum
induced lysis of M. lysodeikticus suspensions lysozyme in patients with renal disease. N. Engi. J. Med. 270, 269
0, control, nolysozyme; , 0, and 0, have added 0.1, 0.5, and 1.0mg of ly- (1964).
sozymeper liter, respectively. Open and closed symbols represent independent 8. Klockars, M., Reitamo, S., Weber, T., and Kerttula, Y., Cerebro-
experiments spinal fluid lysozyme in bacterial and viral meningitis. Acta Med.
Scand. 203,71(1978).
suiting in killed or otherwise damaged cell suspensions. 9. Glynn, A., Lysozyme: Antigen, enzyme and antibacterial agent.
In The Scientific Basis of Medicine Annual Reviews, Athlone Press,
Boasson (10) used phenol-treated cells, which proved to be
London, 1968, p 31.
extremely variable. Others used dry cells, with or without prior
10. Boasson, E. H., On the bacteriolysis by lysozyme. J. Immunol.
phenol treatment, or ultraviolet-treated cells. All these 34, 281 (1938).
treatments of the cells caused a decrease in their lytic response
11. Gorin, G., Wang, S. F., and Papapavlou, L., Assay of lysozyme by
(12), presumably because of effects on the lipidor proteins its lytic action on M. lysodeikticus cells. Anal. Biochem. 39, 113
adjacentto the cellwall.Lyophilizedcells, which are quite (1971).
tediousto produce,alsoprovedunsuitable, becauseextensive 12. Smolelis, A. N., and Hartsell, S. E., Factors affecting the lytic
autolysis occurredduringresuspension(11). activity of lysozyme. J. Bacteriol. 63,665 (1952).

CLINICAL CHEMISTRY, Vol. 25, No. 3, 1979 485