The lactic dehydrogenase (LDH) activity of the aqueous humour has been estimated in both eyes of 7 patients having uniocular retinoblastoma. In 1 patient the aqueous humour LDH activity in the healthy eye was above normal, but there was no clinical evidence of malignancy. Tumour tissue was detected in this eye 9 months later, and the aqueous humour showed a rise in LDH activity. A high LDH activity persisted even after irradiation, though no tumour tissue was visible ophthalmoscopically. It is suggested that the estimation of the LDH activity in the aqueous humour of the healthy eye in cases of uniocular retinoblastoma might be of value in the early detection of a bilateral retinoblastoma.
LDH activity was determined in aqueous humour samples from 11 eyes (of 10 children), four of which contained retinoblastoma. Simultaneous serum LDH levels were also determined in eight of the children. There was no correlation between serum and aqueous humour LDH activity. Total aqueous humour LDH activity ranged from 0 to 99 i.u/l. in the seven eyes with non-neoplastic conditions. It was 56, 124, 158, and 1832 i.u./l. respectively, in the four eyes with retinoblastoma. In all four eyes the ratio of isoenzymes LDH5:LDH1 was greater than 5. The total aqueous humour LDH levels in retinoblastoma was neither consistently elevated, nor related to the total serum LDH. There was a characteristic LDH isoenzyme fractionation pattern which, it is suggested, may be present before the total aqueous humour LDH becomes elevated.
An analysis of the lactic acid dehydrogenase (LDH) activity in the aqueous humour of 24 enucleated eyes with retinoblastoma showed that though there was no relationship between the LDH levels and the age of the patient, there appeared to be an increase in the LDH activity with increase in the duration of the tumour. Undifferentiated tumour cells and tumour cell necrosis seemed to contribute to an increase of LDH levels in the aqueous humour, but there was no relationship between the occurrence of calcification and the LDH secretion into the aqueous humour. Massive cell necrosis caused by radiotherapy and central retinal artery occlusion significantly increased the LDH levels in the aqueous. It also appeared that recurrence was common after external cobalt therapy and that secondary extension of the tumour into the optic nerve and choroid was favoured by this procedure.
The isoenzyme pattern of enolase was examined in the aqueous humour and serum of patients with retinoblastoma (10 aqueous, 8 sera), malignant melanoma (4 aqueous, 25 sera), and normal subjects undergoing cataract surgery (25 aqueous, 30 sera). The assay we used allowed assessment of all three major isoenzymes, including the gamma gamma isoenzyme (neurone-specific enolase). No enolase was detectable in normal aqueous; alpha alpha isoenzyme was present in the aqueous of one patient with malignant melanoma, while aqueous from all patients with retinoblastoma contained both alpha alpha and gamma gamma. Normal serum contained only an alpha alpha band, while serum from patients with retinoblastoma contained alpha alpha, alpha gamma, and gamma gamma bands (7 sera, 87.5%), or alpha alpha only (1 patient, 12.5%). All sera from patients with malignant melanoma contained the alpha alpha band, with low levels of gamma gamma in 16 (60%). In a single patient with Coats's disease alpha alpha was present in the serum, but no enolase was detected in aqueous. Increased amounts of gamma-containing isoenzymes of enolase are found in both serum and aqueous from patients with retinoblastoma. In malignant melanoma there is often an increase in serum gamma gamma enolase. The assessment of aqueous and serum enolase patterns may be of value in the diagnosis of retinoblastoma and malignant melanoma.
Normal aqueous humour and the aqueous humour of patients with cataract is virtually protein-free. Patients having retinoblastoma and non-malignant postinflammatory lesions show significantly high quantities of proteins in the aqueous humour. Retinoblastoma is associated with an increase in the globulin content and an albumin/globulin ratio below unity, while non-malignant postinflammatory intraocular conditions show a rise of the albumin fraction with an albumin/globulin ratio above unity. It seems likely that the leakage of protein into the aqueous is different in the two conditions, and a transcellular route is postulated as being the cause in malignant conditions. The estimation of protein patterns in the aqueous humour may be of value in the diagnosis of intraocular malignancy.
The sodium and potassium concentrations and the lactic dehydrogenase (LDH) activity were estimated in the aqueous and serum of five children with retinoblastoma. Two patients who had a high aqueous LDH activity also showed grossly elevated potassium levels in their aqueous. Though the exact reason for this high potassium concentration in the aqueous is not apparent, it seems likely that cell necrosis within the tumour mass was a contributory factor. It is unlikely that aqueous electrolyte estimations in retinoblastoma could prove to be of any diagnostic value.
4-Hydroxy-3-methoxymandelic acid (HMMA) concentrations in aqueous humour, urine, and serum were simultaneously estimated to determine if these levels were raised in cases of retinoblastoma. The catecholamine content of aqueous humour was significantly higher than that of urine and serum, but as there was no significant difference in the HMMA concentration in retinoblastoma and other non-malignant conditions it seems likely that retinoblastoma is not a catecholamine-secreting tumour.
The role of estimating the serum carcinoembryonic antigen (CEA) in the diagnosis and prognosis of retinoblastoma cases has been evaluated. Although the mean serum CEA titre in children with retinoblastoma was higher than that in control children (p = 0.01), the serum CEA level itself was found not to be a useful marker in the diagnosis of retinoblastoma. A significant correlation of serum CEA titre with progression or regression of the disease observed during therapy in most cases indicated that serial assays of serum CEA may be important in the follow-up of cases with retinoblastoma. The lower CEA values in aqueous humour and subretinal fluid from eyes with retinoblastoma than in the serum suggests that the tumour does not secrete the CEA.
The total lactic dehydrogenase (LD) content and LD isoenzyme ratios were studied in homogenized rectal mucosa from 31 patients with established non-specific mucosal ulcerative colitis and from 16 normal subjects. The total LD content was found to be significantly increased in patients with active ulcerative colitis when compared with patients with inactive colitis or with normal subjects. There was a similar though not a significant increase in the slow moving isoenzymes of LD in samples of rectal mucosa from patients with active colitis. The LD isoenzyme pattern was in the normal range in two of the three patients with histological premalignant changes in rectal biopsy specimens.
There was a statistically significant linear correlation between the total lactic dehydrogenase content of rectal mucosa and the carcino-embryonic antigen levels in whole serum.
Both the total content and isoenzyme ratios of LD were increased in neoplastic tissue obtained from patients with carcinoma of the colon and with tissue from benign rectal polyps.
Separation and quantitative estimation of the isoenzymes of lactate dehydrogenase(LD) in serum were accomplished with capillary electrophoresis system. An uncoated fused silica capillary column 50 cm long, 75μm I.D. and substrate containing running buffer including L-lactic acid and NAD+ were used for the separation of serum LD isoenzymes. The resulting product of “NADH” was detected at 340 nm. Injection of 10 nL of five fold diluted serum sample were performed by pressure injection within 2 seconds. The isoenzymes were separated at 10 kV of voltage for 5 min, by turning off the voltage applied for 30 min incubation at 24°C for reaction between substrate and isoenzymes, and applying voltage of 30 min. Under these conditions, the isoenzymes of LD were detected by a NADH generated as isoenzyme of LD-5 emerged at 20 min, LD-1 peak at 23.5 min with close to baseline separation of the other isoenzymes which emerge between LD-5 to LD-1, after the emergence to LD-1 peak, followed another peak, termed “sample shock”: The results obtained by the proposed method correlated well with those by gel electrophoresis systemes (r=0.92∼0.98) for each five LD isoenzymes, respectively. Within-run precision CVs for 5 replicate analysis were 3.01 (LD-3, mean 14.6%)%∼7.82% (LD-4, mean 4.22%.), respectively.
Capillary electrophoresis; LD isoenzymes; analytical method
The isoenzyme pattern of lactic dehydrogenase was measured in rectal biopsies from six patients with ulcerative colitis in whom precancerous histological changes had been independently recognized. There was a highly significant increase in the ratio of isoenzymes IV + V to I + II. This suggests that lactic dehydrogenase isoenzyme measurement may prove a valuable addition to histology in the recognition of precancer in ulcerative colitis.
Eight fermentative mycoplasmas differing in genome size, deoxyribonucleic acid (DNA) base composition, or sterol dependence were examined for lactic dehydrogenase composition by spectrophotometric assay and polyacrylamide gel electrophoresis. Three completely different patterns of lactic dehydrogenase composition were found. (i) A nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactic dehydrogenase was found in Mycoplasma pneumoniae, M. gallisepticum, M. mycoides var. mycoides, mycoplasma UM 30847, M. neurolyticum, and Acholeplasma axanthum. Electrophoresis of cell-free extracts of each of these mycoplasmas produced, with the exception of M. mycoides var. mycoides and UM 30847, single, different enzyme bands. M. mycoides var. mycoides and UM 30847 were similar and formed multiple bands of enzyme activity. We were unable to establish whether these multiple bands were due to lactic dehydrogenase isoenzymes or artifacts. (ii) An NAD-dependent d(−)-lactic dehydrogenase which could not be reversed to oxidize lactate was found in M. fermentans. (iii) A. laidlawii A possessed an NAD-independent d(−)-lactic dehydrogenase capable of reducing dichlorophenol-indophenol, and an NAD-dependent l(+)-lactic dehydrogenase which is specifically activated by fructose-1,6-diphosphate. Heretofore, this enzyme regulatory mechanism was known to occur only among the Lactobacillaceae. No yeast-type lactic dehydrogenase activity was found in any of the mycoplasmas examined. The stereoisomer of lactic acid accumulated during growth correlated perfectly with the type of NAD-dependent lactic dehydrogenase found in each mycoplasma. The types of lactic dehydrogenase activity found in these mycoplasmas were not related to genome size, DNA base composition, or sterol dependence.
Left-ventricular heart muscle and pectoralis major muscle of the rat were studied to determine the intracellular localization of lactic dehydrogenase (LDH) isoenzymes. Fixation of tissue for 2 hr in 2% buffered formaldehyde provided the best preservation of the ultrastructure and enzyme activity. Total LDH activity was found diffusely in the ground substance of the sarcoplasm and in the mitochondria of the heart muscle. In skeletal muscle a strong reaction was noted in the sarcoplasmic reticulum, and moderate activity was seen in the ground substance of the sarcoplasm and in the mitochondria. Differentiation of the isoenzymes of LDH was accomplished by addition of 4 M urea or application of heat. Heart-type isoenzymes were mainly localized in the mitochondria and sarcoplasm, whereas muscle-type isoenzymes were localized mainly in the sarcoplasmic reticulum of the skeletal muscle. It is speculated that the sarcoplasmic reticulum of the skeletal muscle is the site of anaerobic glycolysis and that the sarcoplasm and mitochondria are involved primarily in aerobic metabolism of pyruvate.
We aimed to investigate activities of metalloproteinases 2 (MMP-2) and MMP-9 in aqueous humour of patients with diabetes mellitus with various stages of diabetic retinopathy.
Material and methods
We included 36 samples of aqueous humour of patients suffering from diabetes mellitus, undergoing routine cataract surgery. Seven of them suffered from proliferative diabetic retinopathy (PDR), 3 had diabetic maculopathy and the remaining 26 had background or minimal background retinopathy only. Metalloproteinases 2 and MMP-9 activities in aqueous humour were measured by gelatin zymography combined with the densitometric imaging system. Total protein content in aqueous humour samples was also assessed.
Metalloproteinases 2 activities were present in almost all samples of aqueous humour (32 of 36) and were 2.6-fold higher in patients who suffered from diabetic ocular complications (p < 0.0001). Activities of MMP-2 correlated well with the duration of the disease (correlation = 0.37, p = 0.03) and tended to correlate with total protein levels in aqueous humour (correlation = 0.43, p = 0.06). Metalloproteinases 9 activities were observed only in 2 of 7 patients with proliferative diabetic disease and the enzyme was absent from aqueous humour samples of patients without proliferative retinopathy.
Increased activities of MMP-2 in aqueous humour of patients with PDR may be related to the disease process and support the hypothesis that MMP-2 may be of particular importance in diabetic retinal neovascularization. MMP-9 may be activated at a certain disease stage only.
metalloproteinase 2; metalloproteinase 9; aqueous humour; diabetic retinopathy; diabetes mellitus; zymography
AIMS--To evaluate the efficacy of biochemical parameters in different fluids in the diagnosis of myocardial infarction of different causes, analysed after death. METHODS--The myoglobin concentration and total creatine kinase (CK) and creatine kinase MB isoenzyme (CK-MB) activities were measured in serum, pericardial fluid, and vitreous humour from seven diagnostic groups of cadavers classified according to the severity of myocardial ischaemia and cause of death. Lactate dehydrogenase (LDH) and myosin were measured only in serum and pericardial fluid, and cathepsin D only in pericardial fluid. Routine haematoxylin and eosin and acridine orange staining were used for microscopy studies of heart tissue. RESULTS--In pericardial fluid there were substantial differences between the different groups with respect to CK, CK-MB, and LDH activities and myosin concentrations. The highest values were found in cases with morphological evidence of myocardial ischaemia. CONCLUSIONS--Biochemical parameters, which reach the pericardial fluid via passive diffusion and ultrafiltration due to a pressure gradient, were thus detectable in this fluid earlier than in serum in cases with myocardial ischaemia. These biochemical parameters may be of use for ruling out myocardial ischaemia in those controversial cases in which reliable morphological findings are lacking.
Usher, D. J., Shepherd, R. J., and Deegan, T. (1974).Thorax, 29, 685-689. Serum lactate dehydrogenase isoenzyme activities in patients with asthma. Increases in the serum activities of several enzymes have been reported in patients with asthma. Liver damage, resulting from altered tensions of oxygen and carbon dioxide in the circulation, has been held to be responsible for the majority of this increase, although it has also been suggested that allergic reactions in the lungs might make some contribution.
This communication describes the application of a more specific enzyme assay, the serum lactic dehydrogenase (LDH) isoenzyme pattern, to asthma patients in an attempt to elucidate the source of the increase in enzyme activity. Raised activities of two isoenzymes, LDH-3 and LDH-5, comprised the bulk of the increase in total LDH activity; in contrast, the activities of LDH-1 and LDH-2 were virtually unaltered. Analysis of the distribution of isoenzyme activity in lung tissue homogenate, coupled with knowledge of that in liver, suggested that both tissues contributed towards the effects observed. It appeared probable that the increment in LDH-3 activity arose from lung involvement, whereas the major portion of the increment in LDH-5 activity was derived from the liver.
Background: Increased levels of lactic dehydrogenase (LDH) in the cerebrospinal fluid (CSF) have been reported in association with several intracranial pathologies. No studies have been performed on patients with Guillain–Barré syndrome (GBS).
Aims: To study LDH isoenzymes in CSF of children with GBS.
Methods: CSF samples collected from nine patients with GBS were analysed for total LDH isoenzymes activity, and compared to samples from 15 patients with normal results.
Results: Mean total LDH activity was 33.33 (6.63) U/l. All patients had significantly increased LDH-3 isoenzyme compared to controls. LDH-3 was the predominant fraction, accounting for more than 50% of total LDH activity and present in more than twice the percentage of LDH-1 or LDH-2. By contrast, in the control group, there were high percentages of mainly LDH-1 and LDH-2.
Conclusions: GBS is apparently associated with a distinct LDH isoenzyme pattern in the CSF. More studies are needed to confirm the rise in LDH-3, as serial CSF analyses are unavailable, and to determine the optimum time of analysis when this finding first becomes detectable.
Fifty consecutive patients undergoing coronary artery bypass grafting for chronic stable angina were assessed by serial electrocardiography, preoperative and postoperative myocardial scanning with technetium-99m pyrophosphate, gated radionuclide ventriculography, and serial measurement of creatine kinase, aspartate aminotransferase, urea stable lactic dehydrogenase, and creatine kinase isoenzyme (MB) to assess the incidence of perioperative myocardial infarction and identify the most appropriate diagnostic techniques. The correlation between myocardial scanning and the measurement of peak enzyme and isoenzyme activity was excellent in the diagnosis of perioperative infarction, although electrocardiography proved less helpful. There appeared to be no advantage in measuring creatine kinase MB rather than the more routinely measured enzymes. There were two deaths and evidence of myocardial infarction in five other patients, an incidence of 14%. Perioperative infarction was associated with a significant reduction in resting ejection fraction in two cases. In those patients without evidence of perioperative infarction the mean increase in ejection fraction of 7.8% was statistically significant.
Fifty consecutive patients, 25 undergoing aortic valve replacement and 25 mitral valve replacement, were studied by serial electrocardiography, preoperative and postoperative technetium-99m pyrophosphate radionuclide scanning, and serial measurement of enzymes (creatine kinase, aspartate aminotransferase, urea stable lactic dehydrogenase) and the MB isoenzyme of creatine kinase to define the incidence of preoperative myocardial infarction and to identify the most appropriate diagnostic techniques. The use of myocardial scanning and measurement of peak enzyme activity proved to be accurate indicators of myocardial infarction, but the electrocardiogram was of limited value. The measurement of creatine kinase MB isoenzyme had no diagnostic advantage over that of the other enzymes. There were two deaths in the series, one due to acute pancreatitis after aortic valve replacement and the other due to myocardial injury after mitral valve replacement. There were four non-fatal myocardial infarctions after aortic valve replacement, giving an incidence of 16%, and none after mitral valve replacement, giving an incidence of 4%.
Background/aim: Endothelin 1 (ET-1) is considered the most potent vasoconstrictor in the body and the eye. This molecule may play a significant role in the pathobiology of exfoliation syndrome (XFS), a disorder characterised by a progressive iris vasculopathy. The purpose of this study was to investigate the concentration of ET-1 in the aqueous humour of cataract patients with and without XFS.
Methods: Aqueous humour samples were obtained from 25 consecutive eyes of 25 cataract patients with XFS and an equal number of age matched controls during phacoemulsification cataract surgery. None of the subjects had elevated intraocular pressure or glaucoma. ET-1 concentration in the aqueous was measured using a specific immunoassay with 100% immunoreactivity for ET-1. Total aqueous humour protein concentration was measured with a microplate Coomassie blue based method and was correlated with ET-1 concentration.
Results: Mean ET-1 concentration in the XFS aqueous samples (4.6 (SD 2.3) pg/ml) was significantly higher than that measured in the age matched control samples (2.8 (SD 1.71) pg/ml); (p = 0.006). Although total protein concentration was significantly elevated in the XFS samples (0.380 (SD 0.159) v 0.279 (SD 0.144) mg/ml in the controls); (p = 0.023), no correlation was found between aqueous ET-1 and total protein concentration (p = 0.730).
Conclusion: The increased concentration of ET-1 in the aqueous humour of XFS patients suggests that ET-1 may play a role in the pathobiology of XFS.
exfoliation syndrome; endothelin; aqueous humour
Both intraocular pressure and aqueous humour turnover rate were determined at intervals over three months in three females in order to investigate whether a correlation existed between these variables and the menstrual cycle. Not only was there a lack of correlation between intraocular pressure or aqueous humour flow rate and menses but intraocular pressure and aqueous humour flow rate were also not related to each other. If pharmacologically administered doses of progesterone or oestrogen influence intraocular pressure, the present data indicate that the effect is probably mediated through effects on the aqueous outflow pathways.
To evaluate whether aqueous humour levels of pigment epithelium‐derived factor (PEDF) are associated with monocyte chemoattractant protein‐1 (MCP‐1) in patients with uveitis.
Aqueous humour levels of MCP‐1 and PEDF were determined by ELISA in 34 uveitis samples and 9 cataract control samples.
Aqueous humour MCP‐1 and PEDF levels were significantly higher in patients with infectious or non‐infectious uveitis than in controls (mean (SD) 32.3 (10.7) ng/ml vs 4.48 (1.10) ng/ml vs 0.47 (0.10) ng/ml, and 8.40 (1.30) μg/ml vs 5.01 (0.92) μg/ml vs 1.32 (0.22) μg/ml, respectively, p<0.001). A positive correlation between PEDF and MCP‐1 was found in patients with uveitis (r = 0.39, p<0.01).
The results demonstrated that aqueous humour levels of PEDF were positively associated with MCP‐1 in patients with uveitis. The present observations suggest that aqueous humour levels of PEDF may be a marker of inflammation in uveitis.
There are several animal studies to suggest that pigment epithelium‐derived factor (PEDF) may exert beneficial effects on diabetic retinopathy and uveitis by acting as an endogenous antioxidant. However, the interrelationship between PEDF and total antioxidant capacity in the human eye remains to be elucidated. In this study, PEDF and total antioxidant levels were determined in the aqueous humour of patients with proliferative diabetic retinopathy (PDR) and uveitis, and the relationship between these two markers was investigated.
Aqueous humour levels of PEDF and total antioxidant capacity were determined by an ELISA system in 34 uveitis and 9 PDR samples.
Aqueous humour levels of PEDF and total antioxidant capacity were significantly lower in patients with PDR than those with uveitis (1.8±0.2 μg/ml vs 6.4±0.8 μg/ml and 0.17±0.03 mmol/l vs 0.85±0.05 mmol/l, respectively, p<0.01). A positive correlation between PEDF and total antioxidant capacity was found in patients with PDR and uveitis (r = 0.33, p<0.05).
This study demonstrated that PEDF levels were associated with total antioxidant capacity in aqueous humour levels in humans. These observations suggest that substitution of PEDF may be a therapeutic target for oxidative stress‐involved eye diseases, especially PDR.
A double blind, prospective study was undertaken to compare aqueous humour penetration of topical 0.3% norfloxacin and 0.3% ciprofloxacin and their effect upon normal eyelid flora in 39 patients undergoing cataract surgery. Lid swabs were taken before and after six 1 hourly applications of single drops of ciprofloxacin or norfloxacin given before surgery. Aqueous humour was aspirated at surgery and antibiotic concentration assayed using high performance liquid chromatography. The mean aqueous humour concentrations were: ciprofloxacin 220 ng ml-1, norfloxacin 140 ng ml-1. Although this difference was not statistically significant (p = 0.112) the trend demonstrated may be relevant clinically, especially considering the greater activity of ciprofloxacin. Both coagulase negative staphylococcal (p = 0.004) and total bacterial (p = 0.019) lid counts dropped sixfold after ciprofloxacin treatment but the smaller reductions noted after norfloxacin application did not achieve statistical significance (p > 0.1). The reduction of external eye flora experienced with ciprofloxacin suggests that this may be a useful presurgical prophylactic agent.
AIMS: The purpose of this pilot study was to test whether the rate of collagen synthesis is measurable in the aqueous humour samples in reoperated and previously unoperated eyes. METHODS: The material consisted of 28 eyes of 27 patients, aged 5 to 82 years, in whom aqueous humour samples were obtained during eye surgery. Fifteen patients had no history of previous eye surgery (control group) while 12 patients were re-operated (study group). The carboxyterminal propeptide of type I procollagen (PICP) and the aminoterminal propeptide of type III procollagen (PIIINP) were measured by specific immunoassays in the aqueous humour samples. RESULTS: The mean concentration of PIIINP in the study group (8.4 (SD 12.5) micrograms/l) was statistically significantly larger than that of the control group (0.4 (0.4) micrograms/l) (p < 0.0037). The respective values for PICP were 98.8 (SD 177.7) micrograms/l in the study group and 0.7 (SD 2.8) micrograms/l in the control group (p < 0.0005). The eyes in the study group which were re-operated within 1 year showed values increased 20-fold compared with the eyes in the control group and those eyes in the study group which had had their previous operation more than a year ago. In three eyes aqueous humour samples were also obtained from the encapsulated Molteno bleb and showed values increased 12-fold compared with those from the anterior chamber. CONCLUSIONS: PICP and PIIINP immunoassays are suitable for measuring the rate of collagen synthesis in the aqueous humour and may be useful in studies on pharmacological modulation of wound healing in glaucoma surgery.