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1.  Prognostic significance of aqueous humour lactic dehydrogenase activity. 
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.
PMCID: PMC1043549  PMID: 476033
2.  Aqueous humour lactic dehydrogenase isoenzymes in 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.
PMCID: PMC1042613  PMID: 1138854
3.  Electrophoretic assessment of aqueous and serum neurone-specific enolase in retinoblastoma and ocular malignant melanoma. 
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.
PMCID: PMC1042154  PMID: 2378858
4.  Correlation of aqueous humour lactic acid dehydrogenase activity with intraocular pathology. 
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.
PMCID: PMC1043550  PMID: 476034
5.  Postinflammatory and malignant protein patterns in aqueous humour. 
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.
PMCID: PMC1043432  PMID: 435425
6.  4-hydroxy-3-methoxymandelic acid (HMMA) excretion in retinoblastoma. 
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.
PMCID: PMC1043277  PMID: 687548
7.  Occurrence and Properties of Lactic Dehydrogenases of Fermentative Mycoplasmas 
Journal of Bacteriology  1972;111(3):633-640.
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.
PMCID: PMC251334  PMID: 4115465
8.  Electrolyte imbalances in the aqueous humour in retinoblastoma. 
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.
PMCID: PMC1040630  PMID: 4005214
9.  Advanced Coats' disease. 
Advanced Coats' disease and retinoblastoma can both present with the triad of a retinal detachment, the appearance of a subretinal mass, and dilated retinal vessels. Thus, even the most experienced observer may not be able to differentiate these entities on ophthalmoscopic findings alone. Coats' disease is the most common reason for which eyes are enucleated with the misdiagnosis of retinoblastoma. Ultrasonography is the auxiliary diagnostic test most easily incorporated into the clinical examination, and can be utilized repeatedly without biologic tissue hazard. Ultrasonically identifiable features allowing differentiation between Coats' disease and retinoblastoma include the topography and character of retinal detachment and presence or absence of subretinal calcifications. Ultrasonography is of lesser use in poorly calcified retinoblastoma and in detecting optic nerve or extraocular extension in heavily calcified retinoblastoma. CT is perhaps the single most valuable test because of its ability to: (a) delineate intraocular morphology, (b) quantify subretinal densities, (c) identify vascularities within the subretinal space through the use of contrast enhancement, and (d) detected associated orbital or intracranial abnormalities. Optimal computed tomographic studies, however, require multiple thin slices both before and after contrast introduction and expose the child to low levels of radiation if studies are repeated periodically. MR imaging is valuable for its multiplanar imaging capabilities, its superior contrast resolution, and its ability to provide insights into the biochemical structure and composition of tissues. It is limited in its ability to detect calcium, which is the mainstay of ultrasonic and CT differentiation. Aqueous LDH and isoenzyme levels were not valuable in distinguishing between Coats' disease and retinoblastoma. The value of aqueous NSE levels in the differentiation of advanced Coats' disease and exophytic retinoblastoma deserves further study. Specimens from patients with intraocular hemorrhage should be viewed cautiously, since erythrocytes contain high levels of enolase. Analysis of subretinal aspirates is an extremely accurate method of confirming the diagnosis of Coats' disease. The key diagnostic findings are the presence of cholesterol crystals and pigment-laden macrophages and the absence of tumor cells on fresh preparations. The technique should be reserved for patients where retinoblastoma has been ruled out by all noninvasive means and massive subretinal drainage is anticipated. The natural progression in advanced Coats' disease is toward the development of a blind, painful eye. Spontaneous regression does rarely occur, and some eyes quietly progress to a phthisical state.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC1298633  PMID: 1808814
10.  Significance of carcinoembryonic antigen in retinoblastoma. 
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.
PMCID: PMC1040308  PMID: 6704360
11.  Intraocular matrix metalloproteinase 2 and 9 in patients with diabetes mellitus with and without diabetic retinopathy 
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.
PMCID: PMC3282515  PMID: 22371774
metalloproteinase 2; metalloproteinase 9; aqueous humour; diabetic retinopathy; diabetes mellitus; zymography
12.  Brain natriuretic peptide: identification of a second natriuretic peptide in human aqueous humour 
AIMS/BACKGROUND—To measure aqueous humour levels of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in humans. To compare peptide levels in glaucomatous and control eyes to test the hypothesis that these peptides are increased in glaucoma. BNP and ANP are cyclic endopeptides whose principal biological effects are natriuresis and vasodilatation. Experimental glaucoma in animal models results in elevated aqueous ANP. Intravenous ANP administration in both animals and humans causes lowering of intraocular pressure (IOP). There are equivocal data to support a role for ANP in IOP regulation in human eyes. There are as yet no published data on BNP in human aqueous humour.
METHOD—This was a case-control study. Cases were primary open angle, pseudoexfoliation, and mixed mechanism glaucoma eyes undergoing trabeculectomy. Controls were cataract extraction eyes. There were 47 trabeculectomy eyes (44 patients) and 47 cataract extraction eyes (46 patients) matched for age, sex, race, systemic medications, and type of anaesthetic. 100-200 µl of aqueous humour were aspirated by paracentesis as the first step in the surgical procedure. Peptide levels were later measured by radioimmunoassay.
RESULTS—The presence of BNP and ANP in human aqueous humour was confirmed. BNP was present in higher concentrations than ANP. BNP levels tended to be greater in control eyes—glaucoma median 56.5 (range 0-3526.5) pg/ml versus control median 65.16 (range 0-1788) pg/ml (Wilcoxon signed rank test p = 0.78). ANP levels tended to be greater in glaucoma eyes than in controls: glaucoma median 3 (range 0-68.5) pg/ml versus control median 0 (range 0-60) pg/ml (Wilcoxon signed rank test p = 0.82). ANP and BNP were log linearly related in both groups (r glaucoma group = 0.961, r control group = 0.894).
CONCLUSION—This is the first report of BNP and ANP in human aqueous humour. Peptide levels did not differ significantly between glaucoma and cataract extraction eyes. A linear relation between log BNP and ANP was found. Further studies are required to clarify the role of these peptides in aqueous humour production and IOP regulation.

 Keywords: brain natriuretic peptide; aqueous natriuretic peptide; aqueous humour; glaucoma
PMCID: PMC1722682  PMID: 9924382
13.  Separation of serum LD isoenzymes by capillary electrophoresis 
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.
PMCID: PMC3453625  PMID: 23105312
Capillary electrophoresis; LD isoenzymes; analytical method
14.  Comparison of Aqueous Humour Concentration after Single High Dose Versus Multiple Administration of Topical Moxifloxacin in Rabbits 
For the prevention of postoperative ocular infections prophylactic topical antibiotics are routinely used. Studies evaluating comparative difference between single dose versus multiple dose administration on aqueous humour concentration of moxifloxacin are lacking. This study compared the aqueous humour concentration of moxifloxacin following its topical administration in rabbit eyes with two dose regimens. Twelve albino rabbits were divided into two groups. In group-1, two drops were administered thrice (total six drops) at 2 min intervals, in both the eyes; in group-2, two drops of moxifloxacin were administered three times a day for three days and also two h before aqueous humour collection i.e. on fourth day. Mean aqueous humour concentrations were calculated and compared using Student's ‘t’ test and P<0.05 was considered significant. Moxifloxacin concentration in aqueous humour in group-1 was 23.79 μg/ml and in group-2 was 42.08 μg/ml. Both dosing regimens produced substantially higher aqueous concentrations than the known minimum inhibitory concentration for most bacteria. Moxifloxacin concentration in aqueous humour with multiple instillations is significantly higher than single instillation (P<0.05), which is adequate to cover ciprofloxacin-resistant gram-negative bacteria. Repeated topical moxifloxacin administration achieved significantly higher aqueous humour concentrations than single administration.
PMCID: PMC4243267  PMID: 25425764
Aqueous humour; MIC90; moxifloxacin; rabbiteye
15.  Atriopeptin, sodium azide and cyclic GMP reduce secretion of aqueous humour and inhibit intracellular calcium release in bovine cultured ciliary epithelium 
British Journal of Pharmacology  1999;127(6):1438-1446.
This study examined the involvement of cyclic GMP, protein kinase G and intracellular Ca2+ movements in the modulation of aqueous humour formation.Using the bovine arterially-perfused eye preparation, drug effects on intraocular pressure and aqueous humour formation rate were measured by manometry and fluorescein dilution, respectively. Drug effects on intracellular [Ca2+] were determined by fura-2 fluorescence ratio technique in non-transformed, cultured ciliary epithelium.Intra-arterial injection of atriopeptin (50 pmol) or sodium azide (10 nmol) produced significant reduction in aqueous humour formation (>38%). This was blocked by selective inhibition (KT-5823) of protein kinase G, but not by selective inhibition (KT-5720) of protein kinase A. Reductions of intraocular pressure produced by atriopeptin or azide were almost completely blocked by KT-5823.ATP (100 μM) caused rapid, transient increase in intracellular Ca2+ followed by a slow decline and prolonged plateau. This response showed concentration-dependent inhibition by atriopeptin, azide or 8-bromo cyclic GMP, and this inhibition of the rapid (peak) Ca2+ increase was enhanced by zaprinast (100 μM; phosphodiesterase inhibitor). KT-5823 blocked the suppression of the peak Ca2+ response but not suppression of the plateau.Arterial perfusion of ATP (0.1–100 μM) produced a concentration-dependent decrease in aqueous humour formation.Aqueous humour formation in the bovine eye can be manipulated through cyclic GMP, operating via protein kinase G. Close parallels appear when Ca2+ movements are modified by similar manipulations of cyclic GMP, suggesting that Ca2+ transients may play an important role in aqueous humour formation and that interplay occurs between cyclic GMP and Ca2+.
PMCID: PMC1760659  PMID: 10455294
Aqueous humour; ATP; atriopeptin; azide; bovine ciliary epithelium; cyclic GMP; intracellular calcium; protein kinase G
16.  Studies of lactic dehydrogenase content in rectal mucosal biopsies 
Gut  1974;15(12):972-976.
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.
PMCID: PMC1413068  PMID: 4448412
17.  Abnormal lactic dehydrogenase isoenzyme patterns in ulcerative colitis with precancerous change 
Gut  1971;12(1):16-19.
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.
PMCID: PMC1411463  PMID: 5543370
18.  Serum lactate dehydrogenase isoenzyme activities in patients with asthma 
Thorax  1974;29(6):685-689.
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.
PMCID: PMC470224  PMID: 4450178
19.  Positive correlation between pigment epithelium‐derived factor and monocyte chemoattractant protein‐1 levels in the aqueous humour of patients with uveitis 
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.
PMCID: PMC1955592  PMID: 17166895
20.  Collagen synthesis activity in the aqueous humour of eyes with glaucoma surgery: a pilot study. 
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.
PMCID: PMC505388  PMID: 8664238
21.  Elevated aqueous humour tissue inhibitor of matrix metalloproteinase-1 and connective tissue growth factor in pseudoexfoliation syndrome 
Background/aims: Pseudoexfoliation syndrome (PXF) was recently found to be associated with increased expression of transforming growth factor β1 (TGFβ1) in the aqueous humour. As concern has been raised regarding anti-TGFβ therapy, which can potentially disrupt the maintenance of anterior chamber asso-ciated immune deviation, the authors explored the levels of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF) in aqueous humour to determine if these may represent alternative therapeutic targets.
Methods: Aqueous humour samples were collected from patients who underwent routine cataract surgery. All patients were categorised into three main groups—PXF, uveitis, and control. The PXF group was further subcategorised into three grades based on the density of the exfoliative material observed on biomicroscopy, as well as the presence or absence of glaucoma. TIMP-1, MMP-9, and CTGF levels were measured using specific enzyme immunoassays (ELISA).
Results: Eyes with PXF had significantly higher aqueous humour TIMP-1 concentration (n = 56, mean (SE), 9.76 (1.10) ng/ml) compared with controls (n = 112, 5.73 (0.43) ng/ml, p<0.01). Similarly, the CTGF level in PXF eyes (n = 36, 4.38 (0.65) ng/ml) was higher than controls (n = 29, 2.35 (0.46) ng/ml, p<0.05). Further, the CTGF concentration in the PXF glaucoma group is significantly higher compared with PXF eyes without glaucoma (6.03 (1.09) ng/ml v 2.73 (0.45) ng/ml, p<0.01). The MMP-9 levels were low and below detection limit in all PXF and control samples with no statistical difference between groups.
Conclusion: A raised TIMP-1 level and a low MMP-9 level in aqueous humour of PXF eyes may imply a downregulation in proteolytic activity. The increased CTGF concentration supports the proposed fibrotic pathology of PXF. Regulation of MMP/TIMP expression and anti-CTGF therapy may offer potential therapeutic avenues for controlling PXF associated ocular morbidity.
PMCID: PMC1772498  PMID: 15665347
aqueous humour; connective tissue growth factor; extracellular matrix; pseudoexfoliation syndrome; tissue inhibitor of matrix metalloproteinase-1
22.  Nitric oxide proxies and ocular perfusion pressure in primary open angle glaucoma 
Background: To investigate the levels of nitric oxide (NO) markers in plasma and aqueous humour of patients with primary open angle glaucoma (POAG) and their relation to ocular perfusion pressure.
Methods: Cyclic guanosine monophosphate (cGMP) and nitrite (NO2 −) were determined in plasma and aqueous humour of 38 patients with POAG and 46 controls. Blood pressure and IOP were measured to calculate ocular perfusion pressure (PP).
Results: cGMP and NO2 − plasma levels were significantly decreased in glaucoma patients compared with controls (p = 0.001 v p = 0.004). In the aqueous humour of subjects with POAG, cGMP and NO2 − concentrations were also lower than in normal eyes (p = 0.0001 v p = 0.001). There was a linear association between cGMP in plasma and aqueous humour in glaucomas and controls (r = 0.514, p = 0.029 and r = 0.558, p = 0.004) and this relation differed in the two groups (p = 0.003). Considering glaucoma patients with controls, a positive correlation was found between cGMP and PP (r = 0.379, p = 0.01) and between NO2 − and PP (r = 0.339, p = 0.040). The cGMP/PP correlation was of borderline statistical significance in controls (p = 0.050), whereas it did not attain statistical significance in POAG, as well as the association between NO2 − and PP when glaucomas and controls were considered separately.
Conclusions: The authors found alterations of NO markers in the plasma and aqueous humour of glaucoma patients. Primary or secondary impaired NO balance could alter ocular perfusion pressure.
PMCID: PMC1772173  PMID: 15148207
primary open angle glaucoma; nitric oxide; ocular perfusion pressure
23.  Levels of bimatoprost acid in the aqueous humour after bimatoprost treatment of patients with cataract 
To determine the aqueous humour concentration of the acid hydrolysis products of bimatoprost and latanoprost after a single topical dose of bimatoprost 0.03% or latanoprost 0.005% in humans.
Randomised, controlled, double‐masked, prospective study. 48 eyes of 48 patients scheduled for routine cataract surgery were randomised in an 8:2:2 ratio to treatment with a single 30 μl drop of bimatoprost 0.03%, latanoprost 0.005% or placebo at 1, 3, 6 or 12 h before the scheduled cataract surgery. Aqueous humour samples were withdrawn at the beginning of the surgical procedure and analysed using high‐performance liquid chromatography–tandem mass spectrometry.
Bimatoprost acid (17‐phenyl trinor prostaglandin F2α) was detected in aqueous samples at a mean concentration of 5.0 nM at hour 1, 6.7 nM at hour 3 and 1.9 nM at hour 6 after bimatoprost treatment. After latanoprost treatment, the mean concentration of latanoprost acid (13,14‐dihydro‐17‐phenyl trinor prostaglandin F2α) in aqueous samples was 29.1 nM at hour 1, 41.3 nM at hour 3 and 2.5 nM at hour 6. Acid metabolites were below the limit of quantitation in all samples taken 12 h after dosing and in all samples from placebo‐treated patients. None of the samples from latanoprost‐treated patients contained quantifiable levels of non‐metabolised latanoprost. Non‐metabolised bimatoprost was detected in aqueous samples at a mean concentration of 6.6 nM at hour 1 and 2.4 nM at hour 3 after bimatoprost treatment.
Low levels of bimatoprost acid were detected in aqueous humour samples from patients with cataract treated with a single dose of bimatoprost. Latanoprost acid concentrations in samples from patients treated with latanoprost were at least sixfold higher. These results suggest that bimatoprost acid in the aqueous humour does not sufficiently account for the ocular hypotensive efficacy of bimatoprost.
PMCID: PMC1954751  PMID: 17135335
24.  Endothelin-1 concentration is increased in the aqueous humour of patients with exfoliation syndrome 
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.
PMCID: PMC1772069  PMID: 15031170
exfoliation syndrome; endothelin; aqueous humour
25.  Biochemical assessment of acute myocardial ischaemia. 
Journal of Clinical Pathology  1995;48(2):124-128.
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.
PMCID: PMC502376  PMID: 7745110

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