Archives of
Arch. Oto-Rhino-Laryng.,212, 119--125 (1976)
Oto-Rhino-LaryngologY 9 by Springer-Verlag 1976
Certain Oxidative and Hydrolytic Enzymes in the Middle Ear Effusion in Serous Otitis Media* S. K. Juhn 1, J. S. Huff, M. M. Paparella 1 Universityof Minnesota Hospital, Mayo Box 234, Minneapolis, Minnesota 65455, U.S.A. Department of OtolaryngologyUniversity of Minnesota, Medical School Minneapolis, Minnesota 55455, U.S.A.
Summary. Activities of various oxidative (LDH, MDH) and hydrolytic (LAP, alkaline- and acid phosphatase, and lysozyme) enzymes in serous middle ear effusions (MEE) and serum from patients with serous otitis media were studied. The ratio of enzyme activity between MEE and serum (MEE/serum) was greater than one for all enzymes studied indicating a higher activity of these enzymes in MEE than in serum. These findings are consistent with a hypothesis suggesting the release of enzymes from inflammatory processes in the middle ear cavity. These enzymes presumably originate from 1) enzymes normally present in blood, 2) release of enzymes from inflamed middle ear mucosa, 3) release of enzymes from inflammatory cells present in the effusions. A measurement of enzyme activities in middle ear effusions (MEE) from patients with serous otitis media should aid in the understanding of the origin and the mechanism of formation of these effusions. Enzymes are proteins with specific catalytic functions. Degenerative or inflammatory change of the tissue may result in enzymatic alterations in the tissues involved. Furthermore, the enzymatic tissue changes may be reflected in comparable enzyme changes in the body fluids which bathe the tissue. Studies of the enzyme activity of other body fluids have revealed abnormalities that reflect the presence of local or systemic pathology (Brauer et al., 1963; West et al., 1963). In an earlier paper (Juhn et al., 1971), we reported preliminary findings of the activities of four enzymes in MEE. In the present paper, we have expanded our studies of the enzyme activities in serous MEE from patients with serous otitis media and compared the results with similar enzyme studies of the serum from these patients. Material and Methods
Seventy-seven middle ear effusions from patients with otitis media were obtained by aspiration during therapeutic myringotomy.Blood serums were also obtained from the same patients. The analyses were confined to serous MEE which were clear and amber colored. *
This research was supported by a grant from USPHS (NS 07623-08)
120
S.K. Juhn et al.
Lactate dehydrogenase (LDH) was measured spectrophotometrically. LDH converts pyruvate and NADH (reduced nicotineamide dinucleotide) into lactate and NAD. Enzyme activity was measured by the rate of decrease in absorbancy of NADH (Wroblewski and La Due, 1955). Malate dehydrogenase (MDH) converts oxaloacetate and NADH into L-malate and NAD. The activity of this enzyme was measured by the rate of decrease in absorbancy of NADH resulting from this reaction (Bergmeyer and Bernt, 1963). Leucine aminopeptidase (LAP) splits leucine-p-nitranilide into leucine and p-nitraniline. LAP activity was determined by measuring the yellow color formed by p-nitraniline (Szasz, 1967). Alkaline and acid phosphatase were measured color• with p-nitrophenyl phosphate as substrate at a pH of 10.5 and 4.8 respectively (Bessey et al., 1946; Fishman and Lerner, 1953). Phosphatases split p-nitrophenyl phosphate into organic phosphate and p-nitrophenol, which is yellow in alkaline solution. Enzyme activity was measured by the intensity of color formed during 30 rain incubation. Commercially available test kits (Boehringer Mannheim Corp., West Germany) were used in the LDH, MDH, LAP and alkaline and acid phosphatase determinations. Lysozyme activity was determined by the rate of lysis of micrococcus lysodeikticus (Shugar, 1952). A standard suspension of M. lysodeikticus was made in Tris buffer (pH 8.8) to give an optical density of 0.5 at 650 nm. An optical density drop of 0.001 units/min at room temperature was defined as 1 unit of enzyme activity. A Zeiss spectrophotometer (PMQ II) with microcell attachment was used for the spectrophotometric analysis.
Results and Discussion
The numerical values of the results and of the ratio, MEE/serum, are shown in Table 1. It is clear that activities in MEE were higher than activities in serum for all enzymes studied. The ratio of MEE/serum ranged from 1.8 (alkaline phosphatase) to 4.0 (acid phosphatase). Graphical presentation of results are shown in Figure 1.
Lactatate Dehydrogenase Approximately 3.3 times greater activity of LDH was observed in MEE than in serum. LDH is an intracellular enzyme. It acts in the glycolytic pathway and cataTable 1. Enzyme levels in serum and middle ear effusions (MEE) of patients with serous otitis media Enzyme
Serum
MEE
MEE/Serum ratio
Lactate dehydrogenase (mU/ml) Malate dehydrogenase (mU/ml) Leucine aminopeptidase (mU/ml) Alkaline phosphatase (mU/ml) Acid phosphatase (mU/ml) Lysozyme (Unit/ml)
123.2 + 12.3 a (17)b
411.0 • 62.5 (17)
3.3
105.1 _+ 16.8 (11)
334.2 + 91.2 (11)
3.2
13.4 + 1.7 (9)
25.1 •
45.4 •
4.2 (21)
81.0 +_ 7.8 (21)
1.8
14.1 •
1.8 (14)
56.4 + 9.0 (14)
4.0
3320 +_ 1134 (5)
7400 • 1166 (5)
2.2
a Standard error of the mean b Number in parentheses equals the number of samples
4.3 (9)
2.1
Oxidative and Hydrolytic Enzymes in the Middle Ear Effusion
121
MEE
10,000
zo~
..r
iiiii
1 ,OOO
10,000
1,ooo E
p ~ ooi p < o o l
c
E
iiii
I .... 100
p
Arch. Oto-Rhino-Laryng.,212, 119--125 (1976)
Oto-Rhino-LaryngologY 9 by Springer-Verlag 1976
Certain Oxidative and Hydrolytic Enzymes in the Middle Ear Effusion in Serous Otitis Media* S. K. Juhn 1, J. S. Huff, M. M. Paparella 1 Universityof Minnesota Hospital, Mayo Box 234, Minneapolis, Minnesota 65455, U.S.A. Department of OtolaryngologyUniversity of Minnesota, Medical School Minneapolis, Minnesota 55455, U.S.A.
Summary. Activities of various oxidative (LDH, MDH) and hydrolytic (LAP, alkaline- and acid phosphatase, and lysozyme) enzymes in serous middle ear effusions (MEE) and serum from patients with serous otitis media were studied. The ratio of enzyme activity between MEE and serum (MEE/serum) was greater than one for all enzymes studied indicating a higher activity of these enzymes in MEE than in serum. These findings are consistent with a hypothesis suggesting the release of enzymes from inflammatory processes in the middle ear cavity. These enzymes presumably originate from 1) enzymes normally present in blood, 2) release of enzymes from inflamed middle ear mucosa, 3) release of enzymes from inflammatory cells present in the effusions. A measurement of enzyme activities in middle ear effusions (MEE) from patients with serous otitis media should aid in the understanding of the origin and the mechanism of formation of these effusions. Enzymes are proteins with specific catalytic functions. Degenerative or inflammatory change of the tissue may result in enzymatic alterations in the tissues involved. Furthermore, the enzymatic tissue changes may be reflected in comparable enzyme changes in the body fluids which bathe the tissue. Studies of the enzyme activity of other body fluids have revealed abnormalities that reflect the presence of local or systemic pathology (Brauer et al., 1963; West et al., 1963). In an earlier paper (Juhn et al., 1971), we reported preliminary findings of the activities of four enzymes in MEE. In the present paper, we have expanded our studies of the enzyme activities in serous MEE from patients with serous otitis media and compared the results with similar enzyme studies of the serum from these patients. Material and Methods
Seventy-seven middle ear effusions from patients with otitis media were obtained by aspiration during therapeutic myringotomy.Blood serums were also obtained from the same patients. The analyses were confined to serous MEE which were clear and amber colored. *
This research was supported by a grant from USPHS (NS 07623-08)
120
S.K. Juhn et al.
Lactate dehydrogenase (LDH) was measured spectrophotometrically. LDH converts pyruvate and NADH (reduced nicotineamide dinucleotide) into lactate and NAD. Enzyme activity was measured by the rate of decrease in absorbancy of NADH (Wroblewski and La Due, 1955). Malate dehydrogenase (MDH) converts oxaloacetate and NADH into L-malate and NAD. The activity of this enzyme was measured by the rate of decrease in absorbancy of NADH resulting from this reaction (Bergmeyer and Bernt, 1963). Leucine aminopeptidase (LAP) splits leucine-p-nitranilide into leucine and p-nitraniline. LAP activity was determined by measuring the yellow color formed by p-nitraniline (Szasz, 1967). Alkaline and acid phosphatase were measured color• with p-nitrophenyl phosphate as substrate at a pH of 10.5 and 4.8 respectively (Bessey et al., 1946; Fishman and Lerner, 1953). Phosphatases split p-nitrophenyl phosphate into organic phosphate and p-nitrophenol, which is yellow in alkaline solution. Enzyme activity was measured by the intensity of color formed during 30 rain incubation. Commercially available test kits (Boehringer Mannheim Corp., West Germany) were used in the LDH, MDH, LAP and alkaline and acid phosphatase determinations. Lysozyme activity was determined by the rate of lysis of micrococcus lysodeikticus (Shugar, 1952). A standard suspension of M. lysodeikticus was made in Tris buffer (pH 8.8) to give an optical density of 0.5 at 650 nm. An optical density drop of 0.001 units/min at room temperature was defined as 1 unit of enzyme activity. A Zeiss spectrophotometer (PMQ II) with microcell attachment was used for the spectrophotometric analysis.
Results and Discussion
The numerical values of the results and of the ratio, MEE/serum, are shown in Table 1. It is clear that activities in MEE were higher than activities in serum for all enzymes studied. The ratio of MEE/serum ranged from 1.8 (alkaline phosphatase) to 4.0 (acid phosphatase). Graphical presentation of results are shown in Figure 1.
Lactatate Dehydrogenase Approximately 3.3 times greater activity of LDH was observed in MEE than in serum. LDH is an intracellular enzyme. It acts in the glycolytic pathway and cataTable 1. Enzyme levels in serum and middle ear effusions (MEE) of patients with serous otitis media Enzyme
Serum
MEE
MEE/Serum ratio
Lactate dehydrogenase (mU/ml) Malate dehydrogenase (mU/ml) Leucine aminopeptidase (mU/ml) Alkaline phosphatase (mU/ml) Acid phosphatase (mU/ml) Lysozyme (Unit/ml)
123.2 + 12.3 a (17)b
411.0 • 62.5 (17)
3.3
105.1 _+ 16.8 (11)
334.2 + 91.2 (11)
3.2
13.4 + 1.7 (9)
25.1 •
45.4 •
4.2 (21)
81.0 +_ 7.8 (21)
1.8
14.1 •
1.8 (14)
56.4 + 9.0 (14)
4.0
3320 +_ 1134 (5)
7400 • 1166 (5)
2.2
a Standard error of the mean b Number in parentheses equals the number of samples
4.3 (9)
2.1
Oxidative and Hydrolytic Enzymes in the Middle Ear Effusion
121
MEE
10,000
zo~
..r
iiiii
1 ,OOO
10,000
1,ooo E
p ~ ooi p < o o l
c
E
iiii
I .... 100
p
