The Influence of the Cause of Death
and Age on Human Corneal
Metabolism
Claudia Redbrake, Jacob Becker, Sabine Salla,
Rudolf Stollenwerk, and Martin Reim
Purpose. Little is known about the metabolic status of
human corneas before transplantation. The authors attempted to determine the influence of both cause of
death and age on the corneal metabolism.
Methods. Adenosine-triphosphate (ATP), adenosine-diphosphate (ADP), glucose, and lactate were measured
in the stroma and endothelium of 30 human corneas.
The corneas were divided into four groups according
to cause of death and four groups according to age.
Corneas from donors with diabetes were excluded.
Results. Corneas from patients who died suddenly—because of cardiac infarction, for example—have good
metabolic status even 24 hours after death. In corneas
of patients with cancer or sepsis, the metabolism has
run down. In comparison, corneas from patients with
cancer are in better metabolic condition than those
from donors with sepsis because they have adapted to
catabolism. Corneas donated from patients with renal
insufficiency show results somewhere in between. Statistical evaluation revealed significant differences in ATP
concentrations for corneas from donors who died suddenly and from patients with renal insufficiency compared to corneas from patients with sepsis. It could be
shown that glucose concentrations as a resource for
metabolism increase with age. The best ATP-ADP ratios were found in the group of 40- to 59-year-old donors. Nevertheless, differences between the age groups
were not statistically significant.
discovered about the metabolic changes in human
corneas after death. Only few investigations have been
published by Reim12 and Greiner in the pertinent
literature. Greiner3"5 showed changes in different
phosphates but did not examine the main source of
energy for corneas: glucose. Therefore, the relationship between metabolic changes and the cause of
death on one hand and age on the other have not
been described before for humans.
For transplantation, the most important parts of
the cornea are the stroma and the endothelium because the epithelium usually regenerates. This is why
we measured glucose, lactate, adenosine-triphosphate
(ATP), and adenosine-diphosphate (ADP) in the
stroma and endothelium.
We investigated 30 human corneas to determine
metabolic changes after death to provide some new,
helpful hints for the selection of donor material.
MATERIALS AND METHODS. Materials. Thirty
fresh human corneas from 16 donors were investigated. The corneas were snap-frozen 4.5 to 60 hours
after death. Mean postmortem time was 24.87 hours.
Donor age ranged from 31 to 80 years. Mean donor
age was 60.53 years. The corneas were not suitable for
transplantation because they did not fit the criteria of
our cornea bank, i.e., the donor was younger than 65
years of age or postmortem time was less than 24
hours, although all corneas were clear before freezing.
We did not evaluate the morphology of the endothelium. Corneas were divided into four groups according to cause of death and age. Corneas from patients with diabetes were excluded. Tables 1 and 2
show the mean postmortem time and mean donor age
in the different groups, according to cause of death
and age.
Conclusion. From our results it can be concluded that
Methods. The tenets of the Declaration of Helthe cause of death and systemic metabolism have an
sinki were followed concerning the examination of
influence on corneal metabolism. Results concerning
human material. Informed consent was obtained, as
donor age reflect the well-known fact that donor age
was the approval of the ethical committee of the Techhas no influence on the quality of keratoplasty material.
nical University of Aachen. Eyes were enucleated in
Invest Ophthalmol Vis Sci. 1994; 35:3553-3556.
the Department of Pathology. The corpses were usually brought there 1 to 2 hours after death and were
kept there at 4°C. For transport, the eyes were stored
in a moist chamber for an additional 15 minutes. They
Anyone who gathers human corneas for transplantawere prepared immediately before trephanization in
tion is highly interested in the quality of the material,
irrespective of whether the corneas used are fresh or
our laboratory. The corneal epithelium was scraped
organ-cultured. Much is known about the morphology
off. The corneas were trephined from the bulbus with
and cell density of the endothelium, but little has been
a diameter of 12 mm, and the wet weight was determined. The corneas were then immediately snap-frozen between a convex and a concave stamp that had
From the Eye Clinic, Faculty of Medicine, Technical University of Aachen, Germany.
been cooled by liquid nitrogen. The dry weight was
Supported by Deutsche Forschungsgemeinschafi grant Re 152/30-1.
measured after lyophilization. Then corneas were
Submitted for publication January 20, 1993; revised February 2, 1994; accepted
March 15, 1994.
ground in an oscillating mill cooled by liquid nitroProprietary interest category: N.
gen. The powder was suspended in 1 ml of ice-cooled
Reprint requests: Claudia Redbrake, MD, Eye Clinic, Technical University of
Aachen, Pauwelsstraffe 30, W-52057 Aachen, Germany.
0.5 N perchloric acid and centrifuged at 19,000 rpm
Investigative Ophthalmology & Visual Science, August 1994, Vol. 35, No. 9
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, August 1994, Vol. 35, No. 9
3554
TABLE l.
Distribution of Mean Postmortem Time and Donor Age in Different Groups of
Cause of Death
Group 1
(Sudden Death)
Number
Mean PM time (hours)
Mean (±SD) donor age (years)
13
23.96
75.5 ± 27.5
for 20 minutes in a cooled centrifuge to precipitate
and separate the protein fraction. The clear supernatant was neutralized by 10 N KOH to pH 7-7.5. Glucose, lactate, ATP, and ADP were assayed in this supernatant by enzymatic optical methods according to
Reim.6
Statistical evaluation was carried out by an unpaired Student's Z-test.
RESULTS. The highest glucose concentrations
were discovered in corneas from donors who died suddenly (Table 3). Metabolism in these corneas seemed
intact, whereas in corneas from patients with cancer
and sepsis, the metabolism had run down. Corneas
from patients with renal insufficiency were somewhere
in between. Because of the high standard deviation,
differences in glucose values were not statistically significant.
The values for lactate were statistically significant
for corneas from patients with cancer and sepsis compared to corneas from patients with renal insufficiency
(P < 0.05). ATP values of corneas from patients who
died suddenly or because of renal insufficiency were
statistically significant compared to those from patients with sepsis (P < 0.05). No statistically significant
differences were found for ADP.
The best ATP-ADP ratios were found in the 40to 59-year-old group, whereas the highest glucose concentrations and glucose-lactate ratios were found in
the group of 60- to 79-year-old donors (Table 4).
Group 1 (20 to 39 years) shows the result of two corneas from one donor, and group 4 (>80 years) represents only one cornea. These two groups are difficult
to interpret. We could find no statistically significant
differences between the age groups for any of the
parameters.
TABLE 2.
Group 2
(Cancer)
Group 3
(Septicemia)
Group 4
(Renal Insufficiency)
7
21.5
66.7 ± 13.3
6
15.42
48.3 ± 23.7
4
25
75.5 ± 2.5
DISCUSSION. Cause of Death. The highest
amount of glucose was discovered in corneas from
donors who died suddenly. It would appear that a fast
death shows metabolic results similar to the in vivo
status of the cornea, although the mean postmortem
time in this group was about 24 hours. There is also
still enough glucose for use, so that the ATP-ADP
ratio was above 1. Reim found ATP-ADP ratios of
approximately 3 immediately after death in fresh donor corneas. 2 Nevertheless, the metabolic status after
24 hours is satisfactory. These changes are due to both
the stasis of the aqueous humor and to cooling at 4°C.
An ATP-ADP ratio of 3 was also described by Greiner
in corneas of young, healthy donors within 4 hours of
death. 5 Unfortunately, a comparison with the results
presented by Greiner is not possible because his data
are presented in terms of the percentage of the total
phosphorus detected and not as an absolute amount,
as was the case in our findings.
The results are completely different in the groups
with tumor and sepsis. As can be expected, both diseases lead to catabolism, which is reflected in the results for glucose and ATP. Both concentrations are
very low. This shows that the metabolism of the cornea
runs down even more in patients with sepsis than in
patients with cancer. Patients with cancer usually suffer much longer before death than patients with sepsis. The metabolism therefore adapts to the catabolic
situation. This leads to higher glucose levels in patients with tumor than in those with sepsis, whereas
the situation was reversed for lactate concentrations.
Corneas from patients with sepsis appear to metabolize almost everything as soon as possible. This is why
lactate increases and ADP concentrations are approximately 10 times higher than ATP levels. Our suggestion is also supported by the shorter postmortem time
Mean Donor Age and Postmortem Time in Different Age Groups
Group 1
Age (years)
Number
Mean age (years)
Mean (±SD) postmortem time (hours)
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Group 2
Group 3
20-39
40-59
60-79
24 ± 0
50.3
31.33 ± 19.67
71.3
20.27 ± 15.77
2
31
12
15
Group 4
>80
1
80
18 ± 0
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Reports
TABLE 3.
Concentrations of Glucose, Lactate, ATP, and ADP in Relation to Cause of Death
Glucose
Lactate
ATP
ADP
ATP/ADP ratio
Group 1
Group 2
Group 3
Group 4
7.8079 ± 9.9411
5.0584 ± 2.4967
0.0782 ± 0.0450
0.0773 ± 0.0989
1.0118
0.2322 ± 0.2295
5.5627 ± 1.7832
0.0501 ± 0.0527
0.1416 ± 0.1788
0.3539
0.1628 ± 0.1832
7.2072 ± 2.0841
0.0297 ± 0.0254
0.2072 ± 0.1689
0.1434
0.1763 ± 0.3054
2.8629 ± 1.2545
0.1071 ± 0.0436
0.1765 ± 0.1087
0.6010
Values are mean ± SD (jzmol/g dry weight).
of the patients with sepsis (15.82 hours compared to
21.5 hours) for the patients with cancer.
The corneas from patients who died of renal insufficiency are somewhere in between. Glucose and lactate levels are low in this group.
The most striking result is the lowest concentration of lactate in corneas from donors with renal insufficiency. These results were statistically significant
compared with the concentrations in corneas of patients with cancer and sepsis. This can be explained by
the metabolic acidosis that occurs after long periods of
renal insufficiency. These patients generally show a
disturbed glycolysis. Lactate increases during the first
phase of renal insufficiency. Under aerobic conditions, it is metabolized to a greater extent into pyruvate because of the higher concentration of lactate.
This would also explain the best ATP result of all four
groups.
Age of Donor. The age of the donor is not relevant
to endothelial cell density.7 We therefore have to answer the question as to whether donor age influences
the metabolism. It is remarkable that the glucoselactate-ratio increases with age (except in the cornea
of one patient older than 80 years). This may be due
to the slower metabolic processes in older individuals.
Nevertheless, ATP concentrations are almost the
same among the different age groups. The ATP-ADP
ratio is higher in the group of 40- to 59-year-old patients, even though the mean postmortem time in this
TABLE
group was the longest of the four age groups. We
conclude that donor age is a less important factor than
cause of death. Differences between the age groups
were not statistically significant. Nevertheless, the metabolic status of older corneas is better than that of
younger corneas at the same postmortem time because of the slower metabolism. Therefore, older corneas can probably be used for a slighdy longer period
of time after death than younger ones.
We cannot yet say to what extent these metabolic
changes are reversible and how much time this process
needs after keratoplasty or during organ culture. On
the basis of our results, it may be possible to develop
special culture media that provide the cornea with
optimum nutrition no matter what the cause of death.
These points will be of further interest for our investigations.
Key Words
cornea, human, donor, metabolism, biochemistry
References
1. Reim M, Hennighausen U, Hildebrandt D, Maier R. Enzyme activities in the comeal epithelium and endothelium of different species. Ophthalmic Res. 197l;2:171-182.
2. Reim M, Foerster KH, Cattepoel H. Some criteria of
the metabolism in the donor cornea. Excerpta Medica
Amsterdam. 1971; 2:728-733.
3. Graymore CN. The cornea. In: Biochemistry of the Eye.
London: Academic Press; 1970:5-37.
4. Concentrations of Glucose, Lactate, ATP, and ADP in Relation to Donor Age
Group 1
Group 2
Group 3
Group 4
Age (years)
Glucose
20-39
0.1951 ± 0.1951
40-59
2.3640 ± 5.2480
60-79
5.0669 ± 9.2953
Lactate
7.3081 ± 1.6677
6.2584 ± 2.4963
4.1757 ± 1.9239
ATP
0.0621 ± 0.0010
0.0663 ± 0.0379
0.0707 ± 0.5340
ADP
0.1314 ± 0.0546
0.0824 ± 0.0986
0.1326 ± 0.1497
0.4722
0.8049
0.5330
>80
0
0
8.0725
0
0.1511
0
0
0
0
ATP/ADP ratio
Values are mean ± SD (^tmol/g dry weight).
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3556
Investigative Ophthalmology & Visual Science, August 1994, Vol. 35, No. 9
4. Greiner JV, Kopp SJ, Gillette TE, Glonek T. Phosphatic metabolites of the intact cornea by phosphorus31 nuclear magnetic resonance. Invest Ophthalmol Vis
ScL 1983;24:535-542.
5. Greiner JV, Lass JH, Gloner T. Interspecies analysis
of cornealphosphate metabolites. Exp Eye Res. 1989;
49:523-529.
Eye Movement Abnormalities in
Carriers of Blue-Cone
Monochromatism
Irene Gottlob
Purpose. Although impaired color vision and ERG
changes have been detected in carriers of blue-cone
monochromatism (BCM), no eye movement abnormalities have been identified. Quantitative eye movements
of three obligate carriers of BCM were analyzed.
Methods. Horizontal and vertical eye movements of
three obligate carriers of two families with BCM with
visual acuity of 20/20 or better were recorded using the
magnetic search coil technique. Subjects were examined fixing in primary and eccentric gaze and during
horizontal and vertical smooth pursuit at 20°, 40°, and
80° per second.
Results. All carriers displayed fixation instability. In two
subjects, fine-amplitude upbeat, jerk-type nystagmus
was detected. Reduced pursuit gain was found in the
carriers. The third subject had small downbeat nystagmus.
Conclusion. Abnormal eye movements are described for
the first time in carriers of BCM. The nystagmus is
clearly distinct from congenital or latent nystagmus and
is similar to the nystagmus reported in BCM. Because
all carriers had excellent visual acuity, in BCM, nystagmus is intrinsic to the disease and can appear independently of the visual defect. Invest Ophthalmol Vis Sci.
1994; 35:3556-3560.
Blue-cone monochromatism (BCM) is a rare congenital color blindness in which normally functioning
blue cones are believed to be present. Affected males
From the Foerderer Eye Movement Center for Children, Wills Eye Hospital,
Philadelphia; the Department of Ophthalmology, Thomas fefferson University,
Philadelphia, Pennsylvania; and the First University Eye Clinic, Vienna, Austria.
Supported by grant f0603-MED from Erwin-Schrddinger, Vienna, Austria.
Submitted for publication fune 14, 1993; revised March 23, 1994; accepted March
23, 1994.
Proprietary interest category: N.
Reprint requests: Dr. Irene Gottlob, Foerderer Eye Movement Center for Children,
Wills Eye Hospital, 900 Walnut Street, Philadelphia, PA 19107.
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
6. Reim M, Meyer D, Cattepoel H. Uber die Bedeutung
des Frierstopverfahrens fur den in vivo Status der Metabolite im Cornea-epithel. Graefe's Arch ClinExp Ophthalmol 1967; 174:97-102.
7. Stocker FW. The Endothelium of the Cornea and Its Clinical Implications. 2nd ed. Springfield, IL: Charles C.
Thomas; 1971:32-34.
have decreased visual acuity and nystagmus. Yee et
al1 found that patients with BCM have continuous or
intermittent pendular or jerk-type nystagmus that can
be horizontal, vertical, or oblique. Photophobia and
myopia are often present.23 An X-linked recessive pattern of inheritance in the absence of cone function is
a reliable indicator of this disease. Most carriers are
clinically normal but, on careful testing, demonstrate
abnormalities in color vision, delays in dark adaptation, abnormal ERG flicker, and dark-adapted red
flash responses or minor macula changes on fluorescein angiography.2"4
Nystagmus and abnormal eye movements have
been found in female carriers of X-linked congenital
nystagmus.5"8 However, in the investigated female carriers of BCM, no pathologic nystagmus or abnormal
eye movements have been detected.1'4 It is unclear
whether in BCM nystagmus is intrinsic to the disease
and if it can appear independently of the visual defect.
We describe three female carriers from two different families with BCM who displayed abnormalities
on quantitative eye movement recordings.
PATIENTS AND METHODS. The research fol-
lowed the tenets of the Declaration of Helsinki. All
subjects gave informed consent, and the study was
approved by the Institutional Review Board Committee of Wills Eye Hospital. Three patients and three
obligate carriers of two families with X-linked inheritance pattern of color blindness, reduced visual acuity,
and nystagmus were included in this study. In family
A, examined carriers included the grandmother (47
years of age) (subject Al) and mother (28 years of
age) (subject A2) of a 10-year-old boy with BCM. The
carrier examined in family B (44 years of age) (subject
B) was the mother of two boys (14 and 9 years of age)
with BCM. Clinical examinations, electroretinograms,
and color tests (Berson color plates, 100 Hue test, and
Panel D 15 test) of patients were consistent with BCM.
Both boys in family B had esotropia. Affected subjects
had predominantly oblique intermittent pendular nystagmus on clinical examination and eye movement
recordings (electrooculograms). Small fixation insta-