Albrecht v. GraefesArch. klin. exp. Ophthat. 201, 263--266 (1977)
Graefes Archiv ffir klinischeundexperimentelle
Ophthalmologie 9 by Springer-Verlag 1977
The Role of Lactic Acid in Neovascularizations Gy6rgy
I mre
Second Department of Ophthalmology, SemmelweisMedical University, Maria u. 39, 1085 Budapest, Hungary
Summary. In this study the aqueous lactate concentration was determined. There was a significant increase of the aqueous lactate content in cases of rubeosis iridis and in cases of large intraocular malignant melanoma where a rubeosis iridis was present in 22%. These data seem to confirm the importance of the increased lactate concentration in neovascularizations.
Zusammenfassung.In dieser Arbeit wurde die Laktatkonzentration des Kammerwassers bestimmt. Eine signifikante Steigerung der Laktatkonzentration des Kammerwassers wurde bei Rubeosis iridis und bei grogen Aderhautmelanoblastomen gefunden. 22 Prozent der Augen mit grogen Tumoren hatten eine Rubeosis iridis. Diese Angaben best~itigen die wichtige Rolle der erh6hten Laktatkonzentration in Gef~igneubildungen. In a previous paper we have suggested on the basis of clinical and experimental data that the prolonged increase in the lactic acid concentration of vascularizing tissues seems to be the most important condition for neovascularization (Imre, 1972). To Gerke et al. (1976) it seems questionable, however, if lactate plays a major role in retinal neovascularization. They determined the lactate content in vitreous-retina-choroid specimens of 02treated kittens and streptozotocin-diabetic rats and there was no difference of lactate content between these specimens and controls. In order to decide whether the lactate has any rolein the mechanism of neovascularizations, it is really reasonable to examine the lactate content of vascularizing tissues, but there are only few such data available. Levene et al. (1963) induced corneal swelling and vascularization in rabbits, and in the initial stage of corneal vascularization the lactate content of the outer rim of the cornea increased on a dry weight basis from the normal 2.9 to 5.5/~g/mg. Chernova (1968) treated rabbit eyes with 5% sodium hydroxide and found that before corneal vascularization began the lactic acid content of the cornea had significantly increased compared with the control. In rubeosis iridis, the new vessels arise on the surface of the iris. On the basis of this fact, the lactate content of the aqueous humour was determined in cases of intraocular malignant melanoma in which the rubeosis iridis frequently develops, in cases of rubeosis iridis, and in control cases. In this study the results of these examinations are reported.
264
G. Irnre
Material and Methods F r o m the anterior chamber of 44 eyes, aqueous humour was drawn off. In each case o. 1 ml aqueous was mixed immediately with 0.1 ml trichloracetic acid to interrupt the metabolism of the cells possibly present in the aqueous. The lactate concentration was determined by the method of Hullin and Noble (1953) and the significant differences were estimated by means of Student's t-test. Out of 44 eyes, 10 were taken as "healthy controls." In 5 of these eyes, we found the occlusion of one of the branches of the central retinal artery and in the other 5 eyes, advanced glaucoma without any sign of an inflammation. In the second group, there were 27 eyes with intraocular malignant melanoma histologically verified later. In 18 cases there were highly elevated tumors involving at least one-quarter of the choroidal surface. In 9 cases there were only small tumors in the choroid. In these cases the aqueous was drawn off immediately before the enucleation. In the third group, there were 11 eyes with rubeosis iridis. In 4 eyes there were intraocular malignant melanoma; for this reason these cases were included both in the second and in the third group. Rubeosis it• developed in 3 other eyes following the occlusion of the central retinal vein, in 2 eyes due to diabetic retinitis proliferans, and in 2 eyes after some years following an unsuccessful retinal detachment operation. In 5 eyes there was secondary glaucoma, too, as a result of the fibrovascular tissue which filled the chamber angle.
Results The aqueous lactate concentration in the "healthy control" group was 7.68 • 2.08 mM/L (mean -+ SD), in cases of malignant melanoma 11.7 -+ 3.82 mM/L, where the large t u m o r group contained 12.45 • 3.89 raM/L, the small tumor group 8.58 +- 1.96 raM/L, and in cases of rubeosis iridis it was 10.68 -+ 2.47 mM/L. There was a significant difference between the means of the melanoma and control groups (p < 0.02), between the rubeosis and control groups (p < 0.01), between the large and small tumor groups (p < 0.01), and between the large t u m o r and control groups (p < 0.01).
Discussion Physiologically, the lactate concentration in human aqueous humour has been found to be 4 . 5 - 8 mM/L (Langham, 1955; de Berardinis and Tier• 1968). The value found in the "healthy control" group of this study is corresponding. The significantly high aqueous lactate concentration found in the cases of malignant melanoma is almost certainly the result of the increased aerobic glycolysis of the malignant tumors. The similarly significant increase of the aqueous lactate concentration in cases of rubeosis iridis is probably the result of the extensive and prolonged hy-. poxic states of the retina. The lactate obviously diffuses through the vitreous into the
The Role of Lactic Acid in Neovascularizations
265
aqueous, and Ashton (1957) and Smith (1961) have already suggested that the vasoformative factor can reach the aqueous humour in this way, causing neovascularization on the surface of the iris. The aqueous lactate concentration can definitely increase only if the rate of aqueous flow decreases, and, according to our prevous investigations, in cases of rubeosis iridis the rate of aqueous flow is really decreased (Imre, 1970). These data speak for the important role of the increased aqueous lactate concentration in rubeosis iridis, owing to which new vessels will develop in the first place on the border of the pupilla and in the chamber angle, i.e., on places which are in contact with the whole amount of flowing aqueous. According to the investigations of Cappin (1973) there is a significant connection between large intraocular tumors and rubeosis iridis. In our large tumor group, too, there was a rubeosis iridis in 4 cases, i.e., in 22% present, whereas in the small tumor group there was no case with rubeosis iridis. Furthermore, in the large tumor group the aqueous lactate concentration was increased and differed significantly from the value of the small tumor and control groups. Circulatory disorders in the iris are also necessary for the development of the rubeosis (Raitta and Vannas, 1969), and, because they were supposed to be still absent in most of the cases in our large tumor group, rubeosis had not been developed in these cases, in spite of the increased aqueous lactate concentration. It is also probable that a more increased lactate concentration gives rise to neovascularization, but the new vessels may remove a part of the aqueous lactate, as, physiologically, one part of the aqueous lactate is led away through the vessels of the iris (Riley, 1972). Therefore, the aqueous lactate concentration was somewhat decreased in our rubeosis group compared with the large tumor group. The results of this study speak for the important role of the increased lactate concentration in neovascularizations. Therefore, it is worth considering whether the results of the investigations o f Gerke et al. (1976) make this role of lactate really questionable. At the beginning of the proliferative phase of experimental fibroplasia, an increased lactate concentration is only probable in the inner layers of the retina. So the findings of Gerke et al. (1976) prove that at the beginning of this phase the lactate content of the inner layers of the retina, i.e., of the least part of the investigated specimens, did not rise to a degree which could have significantly increased the lactate concentration of the whole vitreous-retina-choroid specimen. On the other hand, the lactate content of the diabetic whole retina could not be found to have increased, even when it was examinated separately. An increased lactate concentration is only probable on the border of areas of capillary occlusions, and where the malnutrition caused by capillary occlusions results in cell destruction, even a low lactate concentration is possible. It is also probable that in the retina of experimental diabetic animals the vasoformative factor can not even be locally accumulated to such an extent that neovascularization is caused, as there are no real neovascularizations in the retina of experimental diabetic animals. Even if the lactate accumulates locally in hypoxic areas to such a degree that it diffuse into the vitreous, there will be a definitely increased lactate concentration causing neovascularization only in the vicinity of the retina. The lactate diffusing further on will be present in the vitreous in more and more diluted concentrations. In this connection, it must be mentioned that in cases of malignant melanoma the lactate diffuses from the whole surface of the tumor into the vitreous, and the vitreous
266
G. Imre
lactate c o n c e n t r a t i o n will be increased b u t n o t locally to a higher degree, so in cases o f intraocular t u m o r , retinal neovascularizations are very rarily seen. In m y o p i n i o n , t h e investigations o f Gerke et al. (1976) do n o t disprove t h e imp o r t a n t role o f increased lactate c o n c e n t r a t i o n in neovascularizations, while our p r e s e n t investigations seem t o c o n f i r m it.
References Ashton, N.: Retinal vascularization in health and disease. Amer. J.Ophthal. 44, 7 - 1 7 (1957) de Berardinis, E., Tieri, O.: Certains problemes qui regardent la composition chimique de l'humeur aqueuse humaine. In: Biochemistry of the eye, symp. 1966. Dardenne, M.V. and Nordmann, J. eds. Basel - New York: Karger 1968 Cappin, J.M. : Malignant melanoma and rubeosis iridis. Brit. J. Ophthal. 57, 815-818 (1973) Chernova, A.A.: The effect of some conservative methods of treatment on the lactic acid content in the alkali burnt cornea. Vesm. Offal. 81, 2 8 - 3 2 (1968) Gerke, E., Spitznas, M., Brodde, O.E.: The role of lactic acid in retinal neovascularization. Albrecht v. Graefes Arch. klin. exp. Ophthal. 200, 79-84 (1976) Hullin, R.P., Noble, R.L.: The determination of lactic acid in microgram quantities. Biochem. 55, 289-291 (1953) Imre, G.: Rubeosis iridis. Acta med. Acad. Sci. hung. 2 7 , 2 0 5 - 2 0 7 (1970) Imre, G.: Neovascularization of the eye. In: Contemporary Ophthalmology honoring Sir St. DukeElder, Bellows, J.G. ed. Baltimore: William and Wilkins 1972 Langham, M.E.: The nature and origin of the aqueous. I. Its general nature. In: Glaucoma, Symp. 1954, Duke-Elder, S. ed. Oxford: Blackwell 1955 Levene, R., Shapiro, A., Baum, J .: Experimental corneal vascularization. Arch. Ophthal. (Chicago) 70, 242-252 (1963) Raitta, C., Vannas, S.: Fluoresceinangiographic der Irisgefitge nach Zentralvenenverschluf~. Albrecht v. Graefes Arch. klin. exp. Ophthal. 177, 33-38 (1969) Riley, M.V.: Intraocular dynamics of lactic acid in the rabbit. Invest. Ophthal. 11,600-607 (1972) Smith, R.: Neovascularization in ocular disease. Trans. Ophthal. Soc. U. K. 8 1 , 1 6 3 - 1 6 7 (1961) Received December 7, 1976
Graefes Archiv ffir klinischeundexperimentelle
Ophthalmologie 9 by Springer-Verlag 1977
The Role of Lactic Acid in Neovascularizations Gy6rgy
I mre
Second Department of Ophthalmology, SemmelweisMedical University, Maria u. 39, 1085 Budapest, Hungary
Summary. In this study the aqueous lactate concentration was determined. There was a significant increase of the aqueous lactate content in cases of rubeosis iridis and in cases of large intraocular malignant melanoma where a rubeosis iridis was present in 22%. These data seem to confirm the importance of the increased lactate concentration in neovascularizations.
Zusammenfassung.In dieser Arbeit wurde die Laktatkonzentration des Kammerwassers bestimmt. Eine signifikante Steigerung der Laktatkonzentration des Kammerwassers wurde bei Rubeosis iridis und bei grogen Aderhautmelanoblastomen gefunden. 22 Prozent der Augen mit grogen Tumoren hatten eine Rubeosis iridis. Diese Angaben best~itigen die wichtige Rolle der erh6hten Laktatkonzentration in Gef~igneubildungen. In a previous paper we have suggested on the basis of clinical and experimental data that the prolonged increase in the lactic acid concentration of vascularizing tissues seems to be the most important condition for neovascularization (Imre, 1972). To Gerke et al. (1976) it seems questionable, however, if lactate plays a major role in retinal neovascularization. They determined the lactate content in vitreous-retina-choroid specimens of 02treated kittens and streptozotocin-diabetic rats and there was no difference of lactate content between these specimens and controls. In order to decide whether the lactate has any rolein the mechanism of neovascularizations, it is really reasonable to examine the lactate content of vascularizing tissues, but there are only few such data available. Levene et al. (1963) induced corneal swelling and vascularization in rabbits, and in the initial stage of corneal vascularization the lactate content of the outer rim of the cornea increased on a dry weight basis from the normal 2.9 to 5.5/~g/mg. Chernova (1968) treated rabbit eyes with 5% sodium hydroxide and found that before corneal vascularization began the lactic acid content of the cornea had significantly increased compared with the control. In rubeosis iridis, the new vessels arise on the surface of the iris. On the basis of this fact, the lactate content of the aqueous humour was determined in cases of intraocular malignant melanoma in which the rubeosis iridis frequently develops, in cases of rubeosis iridis, and in control cases. In this study the results of these examinations are reported.
264
G. Irnre
Material and Methods F r o m the anterior chamber of 44 eyes, aqueous humour was drawn off. In each case o. 1 ml aqueous was mixed immediately with 0.1 ml trichloracetic acid to interrupt the metabolism of the cells possibly present in the aqueous. The lactate concentration was determined by the method of Hullin and Noble (1953) and the significant differences were estimated by means of Student's t-test. Out of 44 eyes, 10 were taken as "healthy controls." In 5 of these eyes, we found the occlusion of one of the branches of the central retinal artery and in the other 5 eyes, advanced glaucoma without any sign of an inflammation. In the second group, there were 27 eyes with intraocular malignant melanoma histologically verified later. In 18 cases there were highly elevated tumors involving at least one-quarter of the choroidal surface. In 9 cases there were only small tumors in the choroid. In these cases the aqueous was drawn off immediately before the enucleation. In the third group, there were 11 eyes with rubeosis iridis. In 4 eyes there were intraocular malignant melanoma; for this reason these cases were included both in the second and in the third group. Rubeosis it• developed in 3 other eyes following the occlusion of the central retinal vein, in 2 eyes due to diabetic retinitis proliferans, and in 2 eyes after some years following an unsuccessful retinal detachment operation. In 5 eyes there was secondary glaucoma, too, as a result of the fibrovascular tissue which filled the chamber angle.
Results The aqueous lactate concentration in the "healthy control" group was 7.68 • 2.08 mM/L (mean -+ SD), in cases of malignant melanoma 11.7 -+ 3.82 mM/L, where the large t u m o r group contained 12.45 • 3.89 raM/L, the small tumor group 8.58 +- 1.96 raM/L, and in cases of rubeosis iridis it was 10.68 -+ 2.47 mM/L. There was a significant difference between the means of the melanoma and control groups (p < 0.02), between the rubeosis and control groups (p < 0.01), between the large and small tumor groups (p < 0.01), and between the large t u m o r and control groups (p < 0.01).
Discussion Physiologically, the lactate concentration in human aqueous humour has been found to be 4 . 5 - 8 mM/L (Langham, 1955; de Berardinis and Tier• 1968). The value found in the "healthy control" group of this study is corresponding. The significantly high aqueous lactate concentration found in the cases of malignant melanoma is almost certainly the result of the increased aerobic glycolysis of the malignant tumors. The similarly significant increase of the aqueous lactate concentration in cases of rubeosis iridis is probably the result of the extensive and prolonged hy-. poxic states of the retina. The lactate obviously diffuses through the vitreous into the
The Role of Lactic Acid in Neovascularizations
265
aqueous, and Ashton (1957) and Smith (1961) have already suggested that the vasoformative factor can reach the aqueous humour in this way, causing neovascularization on the surface of the iris. The aqueous lactate concentration can definitely increase only if the rate of aqueous flow decreases, and, according to our prevous investigations, in cases of rubeosis iridis the rate of aqueous flow is really decreased (Imre, 1970). These data speak for the important role of the increased aqueous lactate concentration in rubeosis iridis, owing to which new vessels will develop in the first place on the border of the pupilla and in the chamber angle, i.e., on places which are in contact with the whole amount of flowing aqueous. According to the investigations of Cappin (1973) there is a significant connection between large intraocular tumors and rubeosis iridis. In our large tumor group, too, there was a rubeosis iridis in 4 cases, i.e., in 22% present, whereas in the small tumor group there was no case with rubeosis iridis. Furthermore, in the large tumor group the aqueous lactate concentration was increased and differed significantly from the value of the small tumor and control groups. Circulatory disorders in the iris are also necessary for the development of the rubeosis (Raitta and Vannas, 1969), and, because they were supposed to be still absent in most of the cases in our large tumor group, rubeosis had not been developed in these cases, in spite of the increased aqueous lactate concentration. It is also probable that a more increased lactate concentration gives rise to neovascularization, but the new vessels may remove a part of the aqueous lactate, as, physiologically, one part of the aqueous lactate is led away through the vessels of the iris (Riley, 1972). Therefore, the aqueous lactate concentration was somewhat decreased in our rubeosis group compared with the large tumor group. The results of this study speak for the important role of the increased lactate concentration in neovascularizations. Therefore, it is worth considering whether the results of the investigations o f Gerke et al. (1976) make this role of lactate really questionable. At the beginning of the proliferative phase of experimental fibroplasia, an increased lactate concentration is only probable in the inner layers of the retina. So the findings of Gerke et al. (1976) prove that at the beginning of this phase the lactate content of the inner layers of the retina, i.e., of the least part of the investigated specimens, did not rise to a degree which could have significantly increased the lactate concentration of the whole vitreous-retina-choroid specimen. On the other hand, the lactate content of the diabetic whole retina could not be found to have increased, even when it was examinated separately. An increased lactate concentration is only probable on the border of areas of capillary occlusions, and where the malnutrition caused by capillary occlusions results in cell destruction, even a low lactate concentration is possible. It is also probable that in the retina of experimental diabetic animals the vasoformative factor can not even be locally accumulated to such an extent that neovascularization is caused, as there are no real neovascularizations in the retina of experimental diabetic animals. Even if the lactate accumulates locally in hypoxic areas to such a degree that it diffuse into the vitreous, there will be a definitely increased lactate concentration causing neovascularization only in the vicinity of the retina. The lactate diffusing further on will be present in the vitreous in more and more diluted concentrations. In this connection, it must be mentioned that in cases of malignant melanoma the lactate diffuses from the whole surface of the tumor into the vitreous, and the vitreous
266
G. Imre
lactate c o n c e n t r a t i o n will be increased b u t n o t locally to a higher degree, so in cases o f intraocular t u m o r , retinal neovascularizations are very rarily seen. In m y o p i n i o n , t h e investigations o f Gerke et al. (1976) do n o t disprove t h e imp o r t a n t role o f increased lactate c o n c e n t r a t i o n in neovascularizations, while our p r e s e n t investigations seem t o c o n f i r m it.
References Ashton, N.: Retinal vascularization in health and disease. Amer. J.Ophthal. 44, 7 - 1 7 (1957) de Berardinis, E., Tieri, O.: Certains problemes qui regardent la composition chimique de l'humeur aqueuse humaine. In: Biochemistry of the eye, symp. 1966. Dardenne, M.V. and Nordmann, J. eds. Basel - New York: Karger 1968 Cappin, J.M. : Malignant melanoma and rubeosis iridis. Brit. J. Ophthal. 57, 815-818 (1973) Chernova, A.A.: The effect of some conservative methods of treatment on the lactic acid content in the alkali burnt cornea. Vesm. Offal. 81, 2 8 - 3 2 (1968) Gerke, E., Spitznas, M., Brodde, O.E.: The role of lactic acid in retinal neovascularization. Albrecht v. Graefes Arch. klin. exp. Ophthal. 200, 79-84 (1976) Hullin, R.P., Noble, R.L.: The determination of lactic acid in microgram quantities. Biochem. 55, 289-291 (1953) Imre, G.: Rubeosis iridis. Acta med. Acad. Sci. hung. 2 7 , 2 0 5 - 2 0 7 (1970) Imre, G.: Neovascularization of the eye. In: Contemporary Ophthalmology honoring Sir St. DukeElder, Bellows, J.G. ed. Baltimore: William and Wilkins 1972 Langham, M.E.: The nature and origin of the aqueous. I. Its general nature. In: Glaucoma, Symp. 1954, Duke-Elder, S. ed. Oxford: Blackwell 1955 Levene, R., Shapiro, A., Baum, J .: Experimental corneal vascularization. Arch. Ophthal. (Chicago) 70, 242-252 (1963) Raitta, C., Vannas, S.: Fluoresceinangiographic der Irisgefitge nach Zentralvenenverschluf~. Albrecht v. Graefes Arch. klin. exp. Ophthal. 177, 33-38 (1969) Riley, M.V.: Intraocular dynamics of lactic acid in the rabbit. Invest. Ophthal. 11,600-607 (1972) Smith, R.: Neovascularization in ocular disease. Trans. Ophthal. Soc. U. K. 8 1 , 1 6 3 - 1 6 7 (1961) Received December 7, 1976
