Lactate
Dehydrogenase
Levels and
Isozyme
Patterns
Measurements in the Aqueous Humor and Serum of Retinoblastoma Patients David H. Abramson, MD; Philip A. Piro, MD; Robert M. Ellsworth, MD; F. David Kitchin, MD; Marguerite McDonald, MD
\s=b\ Lactate dehydrogenase (LDH) activity determined in 24 cases of histologically proved retinoblastoma. The mean level was 1,825 IU/L, with a range of 10 to 15,920 IU/L. Twenty-two patients had elevated aqueous humor LDH levels. In two, the aqueous humor level was within the normal range. Student's t testing showed a highly significant difference between cataract and retinoblastoma aqueous humor. The retinoblastoma aqueous humor to blood ratio ranged from 0.045 to 58,529\p=m-\nota good index of retinoblastoma. Isozyme patterns of retinoblastoma aqueous humor were statistically similar to those of normal cataract aqueous humor. The serum LDH level of was
cataract different
patients
was not statistically from that of retinoblastoma patients. The LDH 5/LDH 1 ratio of retinoblastoma aqueous humor ranged from 0.042 to 27.05\p=m-\nota good index of retinoblastoma, as 28 of 34 aqueous samples of cataract patients had LDH 5 > LDH 1. Ten of 13 retinoblastoma patients had aqueous humor LDH 5 > LDH 1. The best index of the presence or absence of retinoblastoma is the total aqueous humor LDH level.
(Arch Ophthalmol 97:870-871, 1979)
serum ma.
In
of
patients
with retinoblasto¬
1971, it was first reported that
retinoblastoma aqueous humor levels elevated.1 Other studies showed that the aqueous humor LDH level in retinoblastoma may vary greatly and occasionally may be normal.-"4 In two studies-:1 with a total of 11 retinoblas¬ toma cases, the aqueous humor levels were higher than the corresponding serum LDH level and the aqueous humor to serum ratio was believed to be a good index of retinoblastoma. When it was shown recently that the aqueous humor LDH levels of "normal eyes" was approximately one fifth that of normal serum, the true impor¬ tance of aqueous humor to serum ratio appeared in doubt.3 Only recently has the normal aque¬ ous humor LDH isozyme pattern been studied.· The normal aqueous humor isozyme pattern is unlikely that of the corresponding serum, and a bloodaqueous humor barrier for LDH appears similar to the blood-CSF barrier." The isozyme pattern of retinoblas¬ toma aqueous humor was the subject of two studies, both prior to the recent study of "normal" aqueous LDH isozymes. In one study (four cases) the were
ratio of LDH 5 to LDH 1 was greater than five." It was not known defini¬ tively if this was characteristic of retinoblastoma aqueous humor or just normal aqueous humor. It is known that the LDH isozyme pattern may be characteristic for many diseases (myocardial infarction, renal disease, pregnancy, etc), and Kabak and Romano believed that this LDH 5/LDH 1 ratio was characteristic of retinoblastoma.1 In contrast, Kaneko4 studied the isozyme pattern in 16 cases of retinoblastoma and found elevations in the LDH fractions 2, 3, and 4; of Kaneko's 16 cases, only two showed detectable LDH 5 levels. Unfortunately, his total levels and
especially isozyme patterns
are
spe¬
cious since he froze his aqueous humor samples after paracentesis, and LDH 5 and LDH 4 are cold unstable and,
therefore,
are
destroyed by freezing.
the purpose of this study to determine the LDH levels in the aque¬ ous and serum samples of retinoblas¬ toma patients and to compare them statistically to normal aqueous humor and serum levels. By avoiding freez¬ ing the samples, prolonged storage, or RBC contamination, we hoped to determine if there is a distinctive It
was
Höhere has been considerable interest and confusion about lactate dehyand isozyme patterns in the aqueous humor and
Table
drogenase (LDH) levels
Accepted for publication Aug 31, 1978. From the Department of Ophthalmology, Edward S. Harkness Eye Institute, ColumbiaPresbyterian Medical Center and Manhattan Eye, Ear, and Throat Hospital, New York. Reprint requests to 70 E 66th St, New York, NY 10021 (Dr Abramson).
1.—Summary
No. of Retinoblastoma Aqueous LDH
Serum LDH
Aqueous/blood ratio Cataract aqueous
Samples 24
21s 21 34
of Data
Range, IU/L 10 170
Mean, IU/L
to
15,920
1,825
to
341
305
0.045 to 58,529 10 167 to
47
•Three hemolysed. tSeven patients had blood greater than aqueous; 14 had aqueous greater than blood.
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/11/2015
Table 2.—Normalized Values of Cataract Aqueous* LDH 5 4 3
2 1
Mean, %t
Range, %t 4.6 0.0 0.0 0.0 0.0
to 100
28.42
to to
31.33 16.70 14.02 8.22
61 33
to
41
to
27
Table 3.—Normalized Values of Retinoblastoma
(Test 7.0 to 30.0 6.4 to 41.7 18.9 to 33.1
(Retinoblastoma/Cataract) .288
18.85 28.33 29.03 17.94 8.42
6.2 to 38.3 1.1 to 25.9
.09 -.21
"Thirteen with isozyme pattern. tValues not significant In all cases.
LDH isozyme pattern for retinoblas¬ toma aqueous humor and to determine how valuable the LDH 5/LDH 1 ratio
Table 4.-LDH 5/LDH 1 Ratios Retinoblastoma 13
No.
isozyme determinations
Aqueous
Cataract Aqueous 34
0.042 to 27.05
Range
MATERIALS AND METHODS
performed on the aqueous and venous blood samples of 24 eyes with presumed retinoblastoma. This technique previously has been reported by us."· The primary aqueous samples (approximately 0.2 mL) were collected under general anesthesia using 30-gauge needles and a 1-mL tuber¬ culin syringe. Using a hemocytometer, cell counts were done on all aqueous samples provided that the quantity was sufficient. The blood samples were spun within two hours of
Mean, %
Range, %
LDH
SD 22.16 16.36 8.21 10.23 6.59
"Thirty-four with isozyme pattern. tValues ranged from 10 to 155 IU/L. tMean value was 47.12 IU/L.
The LDH and
Aqueous*
LDH 5
>
LDH 1
>
LDH 1 LDH 5
28
10(77%) 3 (23%)
6
(82%) (18%)
were
collection; analysis
was
immediately
usually performed
thereafter. Statistical data from aqueous and blood of cataract patients were obtained from our prior study."1 Data are summarized and normalized values are presented in Tables 1 to 4. COMMENT
Twenty-two of 24 patients with histologically proved retinoblastoma had high LDH levels. The mean reti¬
noblastoma LDH aqueous humor level of 1,825 IU/L was statistically signifi¬ cant when compared with cases of
cataract aqueous
(P < .02).
In two
of this series, retinoblastoma eyes had normal levels of aqueous humor LDH. Clearly, all cases of reti¬ noblastoma are not associated with elevated aqueous LDH levels. The false-negative rate for this series was 8.3%. The aqueous to blood ratio of LDH previously has been suggested to be cases
index of the presence of ocular retinoblastoma. Kabak and Romano found that all six of their cases had aqueous/serum levels greater than one.3 The aqueous to blood ratio in our series ranged from 0.045 to 58,529. One third of our cases showed aqueous to blood ratios of less than one. This sharp difference in ratios occurs for a number of reasons. On one hand, in retinoblastoma cases the aqueous lev¬ els themselves may vary more than 1,000-fold. The serum samples may be falsely elevated due to hemolysis, but even if not hemolysed, the mean serum level is approximately four times that of the mean aqueous level in cataract patients. Thus, the aque¬ ous level may be elevated from a tumor and still may be less than the serum LDH level. The aqueous to blood ratio need not be greater than one in the presence of ocular retino¬ blastoma. When statistical analysis of LDH 1, 2, 3, 4, and 5 levels were performed, there was no difference between reti¬ noblastoma and cataract aqueous iso¬ zyme fractions. It has been reported previously that the LDH 5/LDH 1 ratio is greater than five in retinoblastoma.4 We found that this ratio varied from 0.042 to 27.05 and that an
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/11/2015
LDH 5
was
greater than LDH 1 in
only ten cases. It must be remembered that 92% of cataract aqueous samples
have an LDH 5/LDH 1 ratio greater than one, and that the LDH 5/LDH 1 ratio appears to be more indicative of aqueous than of retinoblastoma aque¬ ous
per
se.
The best indication of retinoblasto¬ ma in the eye appears to be the abso¬ lute levels of aqueous LDH when the sample is fresh, not frozen, and free of blood. References 1. Dias PLR, Senthe Shanmuganathau S, M: Lactate dehydrogenase activity of aqueous humor in retinoblastoma. Br J Ophthalmol 55:130-132, 1971. 2. Swartz M, Herbert RW, Goldberg MF: Aqueous lactic acid dehydrogenase in retinoblastoma. Am J Ophthalmol 78:612-617, 1974. 3. Kabak J, Romano PE: Aqueous humor lactic dehydrogenase isoenzymes in retinoblastoma. Br J 59:268-269, 1975. 4. Kaneko A: Lactic acid dehydrogenase activity and isoenzymes in the retinoblastoma. Acta Soc Ophthalmol Jpn 76:672-682, 1972. 5. McDonald MB, Abramson DH, Ellsworth RM, et al: Lactate dehydrogenase levels and isoenzyme patterns in the serum and aqueous humor of adult cataract patients. Arch Ophthalmol 95:2068-2069, 1977. 6. Wroblewski E, Decker B, Wroblewski R: Activity of lactic dehydrogenase in spinal fluid. Am J Pathol 28:269, 1957.
Rajaratnam
Ophthalmol
Dehydrogenase
Levels and
Isozyme
Patterns
Measurements in the Aqueous Humor and Serum of Retinoblastoma Patients David H. Abramson, MD; Philip A. Piro, MD; Robert M. Ellsworth, MD; F. David Kitchin, MD; Marguerite McDonald, MD
\s=b\ Lactate dehydrogenase (LDH) activity determined in 24 cases of histologically proved retinoblastoma. The mean level was 1,825 IU/L, with a range of 10 to 15,920 IU/L. Twenty-two patients had elevated aqueous humor LDH levels. In two, the aqueous humor level was within the normal range. Student's t testing showed a highly significant difference between cataract and retinoblastoma aqueous humor. The retinoblastoma aqueous humor to blood ratio ranged from 0.045 to 58,529\p=m-\nota good index of retinoblastoma. Isozyme patterns of retinoblastoma aqueous humor were statistically similar to those of normal cataract aqueous humor. The serum LDH level of was
cataract different
patients
was not statistically from that of retinoblastoma patients. The LDH 5/LDH 1 ratio of retinoblastoma aqueous humor ranged from 0.042 to 27.05\p=m-\nota good index of retinoblastoma, as 28 of 34 aqueous samples of cataract patients had LDH 5 > LDH 1. Ten of 13 retinoblastoma patients had aqueous humor LDH 5 > LDH 1. The best index of the presence or absence of retinoblastoma is the total aqueous humor LDH level.
(Arch Ophthalmol 97:870-871, 1979)
serum ma.
In
of
patients
with retinoblasto¬
1971, it was first reported that
retinoblastoma aqueous humor levels elevated.1 Other studies showed that the aqueous humor LDH level in retinoblastoma may vary greatly and occasionally may be normal.-"4 In two studies-:1 with a total of 11 retinoblas¬ toma cases, the aqueous humor levels were higher than the corresponding serum LDH level and the aqueous humor to serum ratio was believed to be a good index of retinoblastoma. When it was shown recently that the aqueous humor LDH levels of "normal eyes" was approximately one fifth that of normal serum, the true impor¬ tance of aqueous humor to serum ratio appeared in doubt.3 Only recently has the normal aque¬ ous humor LDH isozyme pattern been studied.· The normal aqueous humor isozyme pattern is unlikely that of the corresponding serum, and a bloodaqueous humor barrier for LDH appears similar to the blood-CSF barrier." The isozyme pattern of retinoblas¬ toma aqueous humor was the subject of two studies, both prior to the recent study of "normal" aqueous LDH isozymes. In one study (four cases) the were
ratio of LDH 5 to LDH 1 was greater than five." It was not known defini¬ tively if this was characteristic of retinoblastoma aqueous humor or just normal aqueous humor. It is known that the LDH isozyme pattern may be characteristic for many diseases (myocardial infarction, renal disease, pregnancy, etc), and Kabak and Romano believed that this LDH 5/LDH 1 ratio was characteristic of retinoblastoma.1 In contrast, Kaneko4 studied the isozyme pattern in 16 cases of retinoblastoma and found elevations in the LDH fractions 2, 3, and 4; of Kaneko's 16 cases, only two showed detectable LDH 5 levels. Unfortunately, his total levels and
especially isozyme patterns
are
spe¬
cious since he froze his aqueous humor samples after paracentesis, and LDH 5 and LDH 4 are cold unstable and,
therefore,
are
destroyed by freezing.
the purpose of this study to determine the LDH levels in the aque¬ ous and serum samples of retinoblas¬ toma patients and to compare them statistically to normal aqueous humor and serum levels. By avoiding freez¬ ing the samples, prolonged storage, or RBC contamination, we hoped to determine if there is a distinctive It
was
Höhere has been considerable interest and confusion about lactate dehyand isozyme patterns in the aqueous humor and
Table
drogenase (LDH) levels
Accepted for publication Aug 31, 1978. From the Department of Ophthalmology, Edward S. Harkness Eye Institute, ColumbiaPresbyterian Medical Center and Manhattan Eye, Ear, and Throat Hospital, New York. Reprint requests to 70 E 66th St, New York, NY 10021 (Dr Abramson).
1.—Summary
No. of Retinoblastoma Aqueous LDH
Serum LDH
Aqueous/blood ratio Cataract aqueous
Samples 24
21s 21 34
of Data
Range, IU/L 10 170
Mean, IU/L
to
15,920
1,825
to
341
305
0.045 to 58,529 10 167 to
47
•Three hemolysed. tSeven patients had blood greater than aqueous; 14 had aqueous greater than blood.
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/11/2015
Table 2.—Normalized Values of Cataract Aqueous* LDH 5 4 3
2 1
Mean, %t
Range, %t 4.6 0.0 0.0 0.0 0.0
to 100
28.42
to to
31.33 16.70 14.02 8.22
61 33
to
41
to
27
Table 3.—Normalized Values of Retinoblastoma
(Test 7.0 to 30.0 6.4 to 41.7 18.9 to 33.1
(Retinoblastoma/Cataract) .288
18.85 28.33 29.03 17.94 8.42
6.2 to 38.3 1.1 to 25.9
.09 -.21
"Thirteen with isozyme pattern. tValues not significant In all cases.
LDH isozyme pattern for retinoblas¬ toma aqueous humor and to determine how valuable the LDH 5/LDH 1 ratio
Table 4.-LDH 5/LDH 1 Ratios Retinoblastoma 13
No.
isozyme determinations
Aqueous
Cataract Aqueous 34
0.042 to 27.05
Range
MATERIALS AND METHODS
performed on the aqueous and venous blood samples of 24 eyes with presumed retinoblastoma. This technique previously has been reported by us."· The primary aqueous samples (approximately 0.2 mL) were collected under general anesthesia using 30-gauge needles and a 1-mL tuber¬ culin syringe. Using a hemocytometer, cell counts were done on all aqueous samples provided that the quantity was sufficient. The blood samples were spun within two hours of
Mean, %
Range, %
LDH
SD 22.16 16.36 8.21 10.23 6.59
"Thirty-four with isozyme pattern. tValues ranged from 10 to 155 IU/L. tMean value was 47.12 IU/L.
The LDH and
Aqueous*
LDH 5
>
LDH 1
>
LDH 1 LDH 5
28
10(77%) 3 (23%)
6
(82%) (18%)
were
collection; analysis
was
immediately
usually performed
thereafter. Statistical data from aqueous and blood of cataract patients were obtained from our prior study."1 Data are summarized and normalized values are presented in Tables 1 to 4. COMMENT
Twenty-two of 24 patients with histologically proved retinoblastoma had high LDH levels. The mean reti¬
noblastoma LDH aqueous humor level of 1,825 IU/L was statistically signifi¬ cant when compared with cases of
cataract aqueous
(P < .02).
In two
of this series, retinoblastoma eyes had normal levels of aqueous humor LDH. Clearly, all cases of reti¬ noblastoma are not associated with elevated aqueous LDH levels. The false-negative rate for this series was 8.3%. The aqueous to blood ratio of LDH previously has been suggested to be cases
index of the presence of ocular retinoblastoma. Kabak and Romano found that all six of their cases had aqueous/serum levels greater than one.3 The aqueous to blood ratio in our series ranged from 0.045 to 58,529. One third of our cases showed aqueous to blood ratios of less than one. This sharp difference in ratios occurs for a number of reasons. On one hand, in retinoblastoma cases the aqueous lev¬ els themselves may vary more than 1,000-fold. The serum samples may be falsely elevated due to hemolysis, but even if not hemolysed, the mean serum level is approximately four times that of the mean aqueous level in cataract patients. Thus, the aque¬ ous level may be elevated from a tumor and still may be less than the serum LDH level. The aqueous to blood ratio need not be greater than one in the presence of ocular retino¬ blastoma. When statistical analysis of LDH 1, 2, 3, 4, and 5 levels were performed, there was no difference between reti¬ noblastoma and cataract aqueous iso¬ zyme fractions. It has been reported previously that the LDH 5/LDH 1 ratio is greater than five in retinoblastoma.4 We found that this ratio varied from 0.042 to 27.05 and that an
Downloaded From: http://archopht.jamanetwork.com/ by a University of Iowa User on 06/11/2015
LDH 5
was
greater than LDH 1 in
only ten cases. It must be remembered that 92% of cataract aqueous samples
have an LDH 5/LDH 1 ratio greater than one, and that the LDH 5/LDH 1 ratio appears to be more indicative of aqueous than of retinoblastoma aque¬ ous
per
se.
The best indication of retinoblasto¬ ma in the eye appears to be the abso¬ lute levels of aqueous LDH when the sample is fresh, not frozen, and free of blood. References 1. Dias PLR, Senthe Shanmuganathau S, M: Lactate dehydrogenase activity of aqueous humor in retinoblastoma. Br J Ophthalmol 55:130-132, 1971. 2. Swartz M, Herbert RW, Goldberg MF: Aqueous lactic acid dehydrogenase in retinoblastoma. Am J Ophthalmol 78:612-617, 1974. 3. Kabak J, Romano PE: Aqueous humor lactic dehydrogenase isoenzymes in retinoblastoma. Br J 59:268-269, 1975. 4. Kaneko A: Lactic acid dehydrogenase activity and isoenzymes in the retinoblastoma. Acta Soc Ophthalmol Jpn 76:672-682, 1972. 5. McDonald MB, Abramson DH, Ellsworth RM, et al: Lactate dehydrogenase levels and isoenzyme patterns in the serum and aqueous humor of adult cataract patients. Arch Ophthalmol 95:2068-2069, 1977. 6. Wroblewski E, Decker B, Wroblewski R: Activity of lactic dehydrogenase in spinal fluid. Am J Pathol 28:269, 1957.
Rajaratnam
Ophthalmol
