Raman Spectroscopy identifies differences in ochronotic and non-ochronotic cartilage; a potential novel technique for monitoring ochronosis.

OBJECTIVE: Alkaptonuria (AKU) is a rare, inherited disorder of tyrosine metabolism, where patients are unable to breakdown homogentisic acid (HGA), which increases systemically over time. It presents with a clinical triad of features; HGA in urine, ochronosis of collagenous tissues, and the subsequent ochronotic arthritis of these tissues. In recent years the advance in the understanding of the disease and the potential treatment of the disorder looks promising with the data on the efficacy of nitisinone. However, there are limited methods for the detection and monitoring of ochronosis in vivo, or for treatment monitoring. The study aim was to test the hypothesis that Raman spectra would identify a distinct chemical fingerprint for the non-ochronotic, compared to ochronotic cartilage. DESIGN: Ochronotic and non-ochronotic cartilage from human hips and ears were analysed using Raman spectroscopy. RESULTS: Non-ochronotic cartilage spectra were similar and reproducible and typical of normal articular cartilage. Conversely, the ochronotic cartilage samples were highly fluorescent and displayed limited or no discernible Raman peaks in the spectra, in stark contrast to their non-ochronotic pairs. Interestingly, a novel peak was observed associated with the polymer of HGA in the ochronotic cartilage that was confirmed by analysis of pigment derived from synthetic HGA. CONCLUSION: This technique reveals novel data on the chemical differences in ochronotic compared with non-ochronotic cartilage, these differences are detectable by a technique that is already generating in vivo data and demonstrates the first possible procedure to monitor the progression of ochronosis in tissues of patients with AKU.

Data on items of AKUSSI in Alkaptonuria collected over three years from the United Kingdom National Alkaptonuria Centre and the impact of nitisinone.

Alkaptonuria is a rare genetic disorder characterized by a high level of circulating (and urine) homogentisic acid (HGA), which contributes to ochronosis when it is deposited in connective tissue as a pigmented polymer. In an observational study carried out by National AKU Centre (NAC) in Liverpool, a total of thirty-nine AKU patients attended yearly visits in varying numbers. At each visit a mixture of clinical, joint and spinal assessments were carried out and the results calculated to yield an AKUSSI (Alkaptonuria Severity Score Index), see "Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre" (Ranganath at el., 2018). The aim of this data article is to produce visual representation of the change in the components of AKUSSI over 3 years, through radar charts. The metabolic effect of nitisinone is shown through box plots.

Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: Evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre.

QUESTION: Does Nitisinone prevent the clinical progression of the Alkaptonuria? FINDINGS: In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period. MEANING: Nitisinone is a beneficial therapy in Alkaptonuria. BACKGROUND: Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU). METHODS: Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at V1 or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre-baseline visit (V0) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended V0, V1, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases. RESULTS: The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/month, in the SAME group, was significantly lower at two (0.32 ±â€¯0.19) and three (0.15 ±â€¯0.13) years post-nitisinone when compared to pre-nitisinone (0.65 ±â€¯0.15) (p < .01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 ±â€¯0.08) and three (0.19 ±â€¯0.06) years post-nitisinone when compared to pre-nitisinone (0.59 ±â€¯0.13) (p < .01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three year's post-nitisinone in both VAR and SAME groups compared with pre-nitisinone (p < .05). CONCLUSION: This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone.

On fragmenting, densely mineralised acellular protrusions into articular cartilage and their possible role in osteoarthritis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26-µm resolution high contrast X-ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re-imaged with 6-µm resolution X-ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self-healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

A systemic provascular response in bone marrow to musculoskeletal trauma in mice.

Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice. Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1(+)) and stem cell factor receptor, CD117 (c-kit(+)) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1(+) mononuclear cell (MNC(sca-1+)) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC(sca-1+,c-kit+) counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours. Sca-1(+) mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1'-dioctadecyl- 3,3,3,'3'-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium. Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures.

Mobilization of endothelial precursor cells: systemic vascular response to musculoskeletal trauma.

Postnatal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells (EPC) migrate, differentiate, and incorporate into the nacent endothelium contributing to physiological and pathological neovascularization, has stimulated much interest. Its contribution to tumor nonvascularization, wound healing, and revascularization associated with skeletal and cardiac muscles ischaemia is established. We evaluated the mobilization of EPCs in response to musculoskeletal trauma. Blood from patients (n = 15) following AO type 42a1 closed diaphyseal tibial fractures was analyzed for CD34 and AC133 cell surface marker expression. Immunomagnetically enriched CD34+ mononuclear cell (MNC(CD34+)) populations were cultured and examined for phenotypic and functional vascular endothelial differentiation. Circulating MNC(CD34+) levels increased sevenfold by day 3 postinjury. Circulating MNC(AC133+) increased 2.5-fold. Enriched MNC(CD34+) populations from day 3 samples in culture exhibited cell cluster formation with sprouting spindles. These cells bound UEA-1 and incorporated fluorescent DiI-Ac-LDL intracellularily. Our findings suggest a systemic provascular response is initiated in response to musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of fracture healing.

PPAR agonists modulate human osteoclast formation and activity in vitro.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.

The impact of ice-skating injuries on orthopaedic admissions in a regional hospital.

Since the opening of a temporary ice-rink in our hospital's catchment area, we have observed an increase in patients requiring in-patient treatment for orthopaedic intervention. The authors performed a prospective analysis of all patients admitted to our unit over a one-month period. Epidemiological data, wearing of protective gear and skater experience were collected. Fracture type, treatment required, average length of hospital stay and number of days missed from work was also recorded. Ice-skating injuries accounted for 7.7% of our total admissions over the study period. There was a significant variation noted in the types of fracture sustained ranging from comminuted fractures of the radial head to spiral fractures of the tibia. The average length of hospital stay was 2.6 days and average time missed from work was 6.1 weeks. This paper highlights the potential serious injuries that can occur in ice-skating and their impact on admissions to our orthopaedic unit.

Volar dislocation of the index carpometacarpal joint in association with a Bennett's fracture of the thumb: a rare injury pattern.

We describe a case of volar dislocation of the index carpometacarpal (CMC) joint in association with a Bennett's fracture of the thumb following a motorcycle accident. Volar dislocation of the index carpometacarpal joint is an exceedingly rare but easily missed injury, with only a few reported cases in the literature. This report highlights the importance of a true lateral radiograph and close scrutiny of the film to detect this injury. Closed reduction supplemented with Kirschner wire fixation restored normal anatomical relations and achieved an excellent clinical result.

Activated protein C attenuates acute ischaemia reperfusion injury in skeletal muscle.

Activated protein C (APC) is an endogenous anti-coagulant with anti-inflammatory properties. The purpose of the present study was to evaluate the effects of activated protein C in the setting of skeletal muscle ischaemia reperfusion injury (IRI). IRI was induced in rats by applying rubber bands above the levels of the greater trochanters bilaterally for a period of 2h followed by 12h reperfusion. Treatment groups received either equal volumes of normal saline or activated protein C prior to tourniquet release. Following 12h reperfusion, muscle function was assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Activated protein C significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet to dry ratio and electrical properties of skeletal muscle. Further in vitro work was carried out on neutrophils isolated from healthy volunteers to determine the direct effect of APC on neutrophil function. The effects of APC on TNF-alpha stimulated neutrophils were examined by measuring CD18 expression as well as reactive oxygen species generation. The in vitro work demonstrated a reduction in CD18 expression and reactive oxygen species generation. We conclude that activated protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury and that this is in part mediated by a direct inhibitory effect on neutrophil activation.

A mechanistic study of the photooxidation of A2E, a component of human retinal lipofuscin.

A major constituent of human retinal lipofuscin is A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-hexatrienyl]-pyridinium). Light transmitted by the lens is absorbed by A2E and the processes initiated by this absorption has been implicated in several maculopothies. The purpose of this study was to evaluate the dominant photochemical mechanisms involved in these reactions, whether through free radical or singlet oxygen intermediacy. The photodestruction of A2E occurs faster in water vs. chloroform and hydrogenated vs. perdeuterated methanol. Both results suggest a free radical mechanism. Product distributions indicate sequential oxygen addition rather than the addition of two oxygen atoms which would be expected if singlet oxygen was an intermediate. Finally, EPR trapping studies lead to the detection of superoxide as the primary intermediate in the photochemical reactions. It is concluded that if singlet oxygen is involved in these photochemical processes it is of minor importance.

Observation of A2E oxidation products in human retinal lipofuscin.

Several retinal dystrophies are associated with the accumulation of lipofuscin in the retinal pigment epithelium (RPE). The only structurally characterized component of human retinal lipofuscin is the bis-retinoid pyridinium compound A2E. We report here on the observation of a monooxygenated product of A2E in the organic soluble portion of human retinal lipofuscin and in the organic extract of bovine RPE cells that have been fed A2E and irradiated. Liquid chromatography mass spectrometry confirms that the products are identical. This is the first observation of a photoproduct of A2E in human retinal lipofuscin.

Release and interconversion of P2 receptor agonists by human osteoblast-like cells.

Nucleotides, acting as agonists at P2 receptors, are important extracellular signaling molecules in many tissues. In bone they affect both bone-forming osteoblast and bone-resorbing osteoclast cell activity. The presence of nucleotides in the extracellular microenvironment is largely determined by their release from cells and metabolism by ecto-enzymes, both of which have scarcely been studied in bone. We have investigated adenosine 5'-triphosphate (ATP) release from SaOS-2 osteoblastic cells and the activities of cell surface ecto-enzymes on ATP metabolism. ATP, but not LDH, was detected in SaOS-2 cell conditioned medium, suggesting these cells were actively releasing ATP. Introduction of ADP resulted in increased ATP concentrations in the medium, which was found not to be receptor mediated. Nucleotide inhibition and substrate specificity studies revealed an ecto-nucleoside diphosphokinase (ecto-NDPK) was responsible for the ADP-->ATP conversion; PCR and immunocytochemistry confirmed its presence. Analysis of ATP metabolism over time demonstrated overall ATP degradation was increased by inhibiting ecto-NDPK activity; confirming that the combined action of multiple osteoblast-expressed ecto-enzymes affected extracellular nucleotide concentration. The data establish the coexistence of ATP-consuming, and for the first time, ATP-generating activities on the osteoblast cell surface, the discovery of which has significant implications for studies involving P2 receptor subtypes in bone.

Arytenoid subluxation.

BACKGROUND: Disruption of the cricoarytenoid joint is uncommon and possibly underdiagnosed. AIM: To highlight the diagnosis of arytenoid subluxation and its management. METHOD: Two case reports. CONCLUSION: Arytenoid subluxation is a rare condition that should always be considered in patients presenting with symptoms following upper airway instrumentation or external trauma to the neck. Early diagnosis optimises the possibility of restoring normal voice and laryngeal function.

Parathyroid hormone-related protein in the aetiology of fibrous dysplasia of bone in the McCune Albright syndrome.

OBJECTIVE: Fibrous dysplasia, observed in bone lesions in the McCune Albright syndrome (MAS), is thought to result from abnormalities in cells of the osteogenic lineage associated with over-activation of the cAMP signalling pathway in affected cells. The aim of this study was to investigate the role of parathyroid hormone-related protein (PTHrP) in the aetiology of MAS, and to determine a possible therapeutic role for 1,25-dihydroxy vitamin D(3) (1,25(OH)(2)D(3)). DESIGN: The effects of 1,25(OH)(2)D(3) on PTHrP production and mRNA expression were determined in vitro. 1,25(OH)(2)D(3) therapy was administered to three patients with MAS. PATIENTS: Clinical data from four MAS patients (MAS1, 2, 3 and 4), and in vitro studies using bone from three MAS patients (MAS1, 2, and 3), are presented. MEASUREMENTS: Immunoradiometric assay and low-cycle number reverse transcriptase-linked PCR were used to determine PTHrP production and mRNA expression in vitro. Standard clinical biochemistry was recorded pre and post commencement of 1,25(OH)(2)D(3) treatment. RESULTS: We report the elevated secretion of PTHrP, and a concomitant rise in PTHrP mRNA expression, in cultured osteoblasts from three MAS patients. Treatment with 1,25(OH)(2)D(3) produced a dose-dependent decrease in PTHrP protein secretion and mRNA expression. Marked improvement in bone biochemistry in MAS1, 2 and 3 post treatment with 1,25(OH)(2)D(3) is documented. CONCLUSION: This study provides the first evidence suggesting that PTHrP may contribute to the aetiology of fibrous dysplasia in MAS. In addition, the therapeutic administration of 1,25(OH)92)D(3) may provide clinicians with an important new regime for symptomatic relief of bone pain and fracture in some patients with MAS.

Regulation of epidermal homeostasis through P2Y2 receptors.

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Human keratinocytes express transcripts for three isoforms of parathyroid hormone-related protein (PTHrP), but not for the parathyroid hormone/PTHrP receptor: effects of 1,25(OH)2 vitamin D3.

Parathyroid hormone-related protein (PTHrP) is strongly expressed in the epidermis and has been implicated in the regulation of growth and differentiation of keratinocytes. PTHrP has N-terminal sequence homology with parathyroid hormone (PTH) and binds to the type I PTH/PTHrP receptor, but earlier reports suggest that keratinocytes do not possess this cell surface receptor. In order to determine which PTHrP mRNA isoforms are expressed by keratinocytes and whether the type I receptor mRNA is present, we designed specific primers for reverse transcriptase-polymerase chain reaction. The interaction of PTHrP with other promoters of keratinocyte differentiation is unclear. In particular, 1,25(OH)2D3 is also fundamental in calcium homeostasis and induces changes in intracellular calcium. We therefore investigated the effect of 1,25(OH)2D3 on PTHrP mRNA expression and protein production in cultured human keratinocytes. Cells were incubated for 3 days at concentrations of 1.25(OH)2D3 of 10(-10)-10(-6) mol/L. PTHrP in culture supernatant, measured by two site immunoradiometric assay, was 915 +/- 98 PTHrP fmol/mg of cell layer protein in untreated cultures decreasing to 570 +/- 113 with 10(-8) mol/L and 402 +/- 24 with 10(-6) mol/L 1,25(OH)2D3 (mean +/- SEM, P < 0.01, n = 6). Transcripts for all three PTHrP isoforms (139, 141 and 173 amino acids) were detectable in keratinocyte mRNA. Corresponding to the decrease in PTHrP protein we demonstrated a reduction in all three PTHrP mRNA transcripts after 3 days' incubation with 1,25(OH)2D3 over a concentration range 10(-10)-10(-6) mol/L. Repeated studies failed to detect type I PTH/PTHrP receptor mRNA in human keratinocytes, either in control cultures or in the presence of 1,25(OH)2D3. We have shown that keratinocytes produce abundant PTHrP and that this is modulated by 1,25(OH)2D3, suggesting a physiological role. Further studies are required to investigate the relative expression of PTHrP isoforms, their role in keratinocyte signalling and the receptors involved.

Measuring oxygen tension in the anterior chamber of rabbits.

PURPOSE: Measuring the concentration of oxygen in the aqueous humor without penetrating the eye would provide a new dimension in understanding aqueous humor and corneal dynamics. In this study a preinvasive method was developed for determining the cameral oxygen concentration in anesthetized rabbits by measuring the excited-state lifetime of a phosphorescent dye. METHODS: A scanning ocular fluorometer was designed to excite phosphorescence with a brief flash of light and to measure the decay of luminescence for as long as 1000 microsec after excitation. The measurement window was scanned through the depth of the anterior chamber or fixed at the mid-anterior chamber. A depot of the phosphorescent dye Pd-uroporphyrin was injected into the vitreous of eight pigmented rabbits, and within a few days the dye was measurable in the anterior chamber. The excited-state lifetime of this dye is inversely correlated to oxygen concentration and was calibrated by measuring the lifetime of dye in cuvettes equilibrated with oxygen-nitrogen mixtures. Oxygen tensions were determined from lifetimes measured in the open eye, under a polymethylmethacrylate (PMMA) contact lens, under two oxygen-permeable contact lenses, and immediately after lid closure. RESULTS: Oxygen tension in the mid-anterior chamber before placing a PMMA contact lens was 23 +/- 3 mm Hg (mean +/- SD; n = 6). After 20 minutes of PMMA lens wear, oxygen tension decreased to 4 +/- 2 mm Hg. When the focal diamond was scanned through the anterior chamber, oxygen tension was 24 +/- 5 mm Hg near the corneal endothelium and decreased to 17 +/- 8 mm Hg near the crystalline lens. Under the PMMA contact lens this gradient reversed: Oxygen tensions near the endothelium and lens were 3 +/- 2 mm Hg and 6 +/- 2 mm Hg, respectively. Lid closure for 10 minutes or longer decreased the mid-anterior chamber oxygen tension from 21 +/- 2 mm Hg (n = 19 measurements from seven animals) to 10 +/- 3 mm Hg (n = 15 measurements from five animals). CONCLUSIONS: Measuring excited-state lifetime of phosphorescent dyes in the anterior chamber provides a useful method for determining oxygen concentration in vivo, without penetrating the eye. Cameral oxygen tension under PMMA contact lenses are significantly lower than in the uncovered eye. The profile of oxygen tension through the anterior chamber suggests that oxygen is supplied transcorneally to the aqueous humor.