Distribution of phosphatic metabolites in the porcine cornea using phosphorus-31 nuclear magnetic resonance

This study describes the phosphatic metabolite contribution of the porcine corneal stroma [by phosphorus-31 nuclear magnetic resonance (P-31 NMR)] and the corneal epithelium (by difference calculation) to the total phosphatic metabolic profile of the intact (i.e. whole, undissected) cornea. In order to provide such a regional analysis, the epithelium and endothelium were surgically removed from three sets of 12 corneas. Three additional sets of 12 intact corneas were used as controls. Histological examination of two randomly selected stromata from each set of corneas were analyzed to verify that epithelium and endothelium were removed. Perchloric acid extracts were prepared from the 10 remaining stromata for P-31 NMR analysis. Twenty phosphatic metabolites were measured including six unidentified (unknown) signals. Components with diminished relative concentrations in the stromal profile compared to that of the intact cornea profile are: the triose and hexose phosphates associated with glycolysis (including alpha-glycerophosphate), choline phosphate, ATP, uridine diphosphohexoses and the unknown compound resonating at 0.93 delta. The relative level of inorganic phosphate in the stroma is nearly doubled. The remaining measured phosphates are not significantly changed relative to the total extractable phosphorus content. Consistent with previous biochemical findings, phosphates associated with energy metabolism are found primarily in the epithelial fraction. The metabolic status of the endothelial monolayer cannot be assessed because of low tissue mass and the relatively low sensitivity of P-31 NMR spectroscopy. This study serves as baseline data for the interpretation of experimental and clinical phosphorus NMR data from intact cornea by providing the contribution to the whole corneal spectrum of metabolites of the individual corneal layers.     

Organophosphate metabolites of the human and rabbit crystalline lens: a phosphorus-31 nuclear magnetic resonance spectroscopic analysis

The concentrations of the major and minor organophosphate metabolites of the rabbit and human crystalline lens were quantitated from perchloric acid extracts analyzed by phosphorus-31 nuclear magnetic resonance spectroscopy. The concentrations of adenosine diphosphate, phosphocreatine, inorganic orthophosphate and the nucleoside diphosphosugars were greater in the human lens samples when compared with the rabbit lens profile. A 10-fold greater phosphocretine content of the human lens was the most remarkable species difference. The adenosine triphosphate and the glycerol derivative, glycerol 3-phosphorylcholine, were enriched in the rabbit lens relative to the human. A prominent resonance signal at 6 p.p.m. corresonding to an as yet, unidentified phosphorus-containing molecule and two other less prominent phosphorus resonances of unidentified origin at 17·9 and 10·7 p.p.m., detected in the rabbit lens were absent from the human lens profile. In addition, the α-glycerophosphate content of the human lens was considerably less than that of the rabbit lens. These profile data document similarities and differences in the human versus the rabbit lens metabolites. The magnitude and diversity of these differences in lens metabolite concentrations emphasize the need for cautious rabbit to human extrapolation of experimental findings.     

Surface coil phosphorus-31 nuclear magnetic resonance studies of the intact eye

The feasibility of employing the surface coil probe technique for the non-invasive study of ocular tissue metabolism by phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) in enucleated bovine, rabbit, human and rat globes is demonstrated. An assessment of individual phosphorus-metabolite contributions from ocular tissues, including the cornea, lens and iris, to the overall 31P NMR spectrum (NMR spectral acquisition parameters optimized for the lens region of the globe) was accomplished through the combination of surgical ablation and difference spectroscopy. The NMR measurements also provided tissue pH values for the lens and cornea. The strengths and limitations of the surface coil NMR method, which is particularly appropriate for in vivo metabolic studies of ocular tissues such as the lens, are discussed.     

Preliminary results on phosphorus-31 nuclear magnetic resonance evaluation of human uveal melanoma in enucleated eyes

Clinical evaluation of uveal melanomas by nuclear magnetic resonance (NMR) techniques depends on ascertaining how these tumors characteristically appear in NMR images and spectra. The authors have determined NMR characteristics of suspected uveal melanomas by phosphorus-31 (31P) NMR spectroscopy of freshly enucleated human eyes. Nuclear magnetic resonance examination was performed at 8.45 Tesla within 90 minutes after enucleation. Enucleated eyes were maintained at 4 degrees C in tissue culture medium during the 30 minutes required for transport. Nuclear magnetic resonance spectra were obtained within 10 minutes, a clinically acceptable time, using a two-turn 31P surface coil. Spectral parameters included 10-kHz spectral width, 1024 data points, and 0.5-second recycle delay. Phosphorus-31 NMR spectroscopy allowed differentiation of choroidal melanomas from normal ocular structures. Differentiating features include significant peaks in tumor spectra due to the phosphodiesters glycerol 3-phosphoryl ethanolamine (GPE) and glycerol 3-phosphorylcholine (GPC), and the phosphomonoesters phosphorylethanolamine (PE) and phosphorylcholine (PC). These preliminary data are encouraging and suggest that clinical trials at the lower magnetic field strengths available in NMR imaging systems seem feasible and warrant investigation.     

Characterization of human uveal melanoma cells by phosphorus 31 nuclear magnetic resonance spectroscopy

We performed experiments to determine the potential usefulness of nuclear magnetic resonance spectra in the diagnosis and follow-up of ocular melanoma. High-resolution phosphorus 31 nuclear magnetic resonance spectra at 109.3 MHz were obtained for human uveal melanoma, Greene hamster melanoma, and normal human diploid fibroblast cells. Phosphate metabolites were identified and their concentrations were shown to vary among the different cell lines. Uveal melanoma cells contain unusually high concentrations of the phospholipid metabolite phosphorylcholine and the phosphodiesters glycerol 3-phosphoryl choline and glycerol 3-phosphoryl ethanolamine. Baseline data are thus provided for studies of the effect of various treatment modalities on uveal melanoma. These initial results suggest that the data provided by high-resolution phosphorus 31 nuclear magnetic resonance spectra can provide useful diagnostic and follow-up data with respect to ocular melanoma.     

Hydration of the animal cornea: spectrometric study using proton nuclear magnetic resonance

The concentration of water in isolated corneas was studied using a Nuclear Magnetic Resonance Protonic (P N M R) technique. The corneas were maintained in moist chambers or in different aqueous fluids in order to compare the results obtained by. P N M R with those provided by classical techniques. The magnetization ratio, associated to water component measured as a function of corneal conservation time has permitted to establish the corneal water content variation. The corneas preserved in moist chamber exhibited a slow dehydration of about 15% in the first day. On the contrary, hydration was evident in liquid media, the strongest in pure water. No significant differences were found between saline, T C Earle medium and Neomycin ophthalmic suspension. P N M R technique give an index of corneal hydration in these conditions and it seems possible to use a P N M R apparatus for the evaluation of a corneal graft before surgery.     

31P magnetic resonance spectroscopy of animal uveal melanoma

Scleral surface coils were used to obtain in vivo magnetic resonance spectra (MRS) of Greene melanoma implanted in the rabbit uvea. Well-localized tumor spectra (4.7 Tesla) with good signal-to-noise ratios (S/N) were obtained from the tumor with a "single-pulse" sequence in less than 1 hour. Tumor localization was confirmed with one-dimensional spectroscopic imaging studies. Serial 31P spectra were obtained during tumor growth and after both optimal and suboptimal hyperthermia. Early 31P MRS change is correlated with tumor treatment response and preceded histologic evidence of cell destruction. Twenty-four to 48 hours after successful treatment, the inorganic phosphate/nucleoside triphosphate (NTP), and phosphomonoester/NTP ratios were significantly increased from 1.2 +/- 0.1 to 1.7 +/- 0.1 and 1.3 +/- 0.1 to 1.8 +/- 0.2, respectively. In contrast, untreated or ineffectively treated tumors showed little change. Interpretation of 31P MRS data in this animal uveal melanoma model after the first week was complicated by decreased S/N, increased contamination from contiguous tissues, ingrowth of fibroblasts, macrophages, and intratumor hemorrhage.     

Preoperative metabolic analysis of donor corneas using magnetic resonance spectroscopy

Successful corneal transplantation was accomplished following metabolic phosphorus magnetic resonance analysis. Four cat corneas were analyzed using phosphorus-31 magnetic resonance following storage in modified McCarey-Kaufman (M-K) medium for 24 h. Corneas were re-stored in M-K medium and transplanted 24 h after MR analysis. Four control corneas (contralateral eye, no magnetic resonance analysis performed) were also transplanted following storage in M-K medium under identical conditions. Successful corneal transplantation was accomplished with minimal ATP tissue levels. Corneas stored for 48 h maintained a pH of 7.3. The phosphorus-31 spectral modulus, which is the ratio of the high-energy phosphates to the low-energy phosphates, was calculated using the spectral integral (range, 0.49-0.77). No difference in endothelial cell density or morphology was detected between corneas following magnetic resonance analysis and control corneas when evaluated by specular microscopy.     

The metabolism of galactose cataracts evaluated by phosphorous-31 nuclear magnetic resonance spectroscopy (2)]

The authors evaluated the metabolic kinetics of organophosphate compounds in the course of the evolution of galactose cataracts in rat lenses using phosphorous-31 nuclear magnetic resonance (31P-NMR) spectroscopy. Three-week-old rats (Lewis) were fed with a 25% galactose diet for three days, and one, two, three and four weeks. Metabolic changes in the lenses were measured and the following results were obtained: (1) Changes in glycerophosphorylcholine (GPC) levels over the time course were analogous to changes for choline phosphate (CP); both GPC and CP level showed a significant decrease in the galactose-fed sample compared with the controls from three days after ingestion, and then continued to decrease gradually. This suggested that the decrease of GPC was associated with the level of CP. (2) Inorganic orthophosphate (Pi) increased gradually after the ingestion of galactose, reaching a maximum at third week, and subsequently falling. Adenosine triphosphate (ATP) levels did not show any significant changes at one week, but had fallen significantly at two weeks. There might be metabolic pathways for production of ATP without requirement of Pi in the lens for at least the first one week. (3) Adenosine diphosphate (ADP) levels did not show any significant changes at three days, but had fallen significantly at one week. Nucleotide sugar (NS) increased gradually after the ingestion of galactose, reaching a maximum at second week, and then decreasing. This suggested that the ADP was converted to ADP sugar.     

31P magnetic resonance spectroscopy (MRS) of experimental orbital myositis

We described a model of orbital myositis that was induced by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) injection into the superior rectus muscle of New Zealand white rabbits. In this study, in vivo 31P magnetic resonance spectroscopy (MRS) was performed with a 4.7 Tesla Oxford (Oxford Instruments, Oxford, England) magnet to monitor the evolution of muscle inflammation in control animals and the response of this model to external beam radiation. 31P MRS showed a dramatic increase in high-energy phosphorus and phospholipid metabolites 48 hr after TPA injection. These spectra were similar to those from implanted allogenic fibroblasts. Within 18-48 hr after a single dose (400 cGy) or sequential doses (3 days at 400 cGy) of orbital irradiation, reduced extraocular muscle swelling and a significant decrease of all 31P metabolites occurred. A decrease (63 +/- 6%) in the signal-to-noise (S/N) ratio of the control inflamed muscle 31P MR spectra increased by 28 days after inflammation. Two days after single-dose radiation, 31P MR metabolites were significantly lower (58 +/- 5%, P less than 0.012) than control spectra. These postradiation spectra mirror the 28-day control spectra and are consistent with previous histologic data that show decreased fibroblastic activity. Change in 31P MRS was a sensitive indicator of treatment response latency.     

Nuclear magnetic resonance spectroscopy

Magnetic resonance spectroscopy is a valuable method for the non-invasive investigation of metabolic processes and can now be combined with conventional magnetic resonance imaging in patients. This article gives a brief introduction into the principles and physiological and clinical applications of in vivo proton magnetic resonance spectroscopy, surveys experiences in healthy volunteers and presents exemplary results in patients suffering from cortical blindness or visual field defects. The causes of visual loss include brain trauma, cerebral ischemia, and brain tumors. In traumatic, ischemic and neoplastic lesions, an important spectral finding is an elevated lactate resonance which has been explained by increased anaerobic glycolysis of ischemic brain tissue and macrophages invading necrotic tissue. In our investigations using a clinical spectroscopy protocol on a 1.5 T MR system, a significant lactate signal was absent in spectra obtained from the visual cortex of normal volunteers, even during photic stimulation with a stroboscope. Other spectral changes in the patients include a decreased N-acetyl-aspartate resonance which indicates a decreased number of viable neurons in the examined brain region.     

In vivo phosphorus 31 magnetic resonance spectroscopy of human uveal melanomas and other intraocular tumors

We studied the feasibility of using the surface coil probe technique for the noninvasive in vivo study of ocular tumors by phosphorus 31 magnetic resonance spectroscopy. The characteristic organophosphate metabolites of suspected uveal melanomas before proton beam irradiation were determined qualitatively by phosphorus 31 magnetic resonance spectroscopy in vivo using a three-turn surface coil. Spectra of choroidal hemangioma, osteoma, and metastasis were also obtained in vivo and compared with those of uveal melanomas. Analysis of spectra performed at 1.5 T showed significant peaks of phosphomonoesters, inorganic phosphate, phosphodiesters, phosphocreatine, and adenosine 5'-triphosphates. The unusually high concentration of phosphodiesters may be considered as a marker for uveal melanomas and other choroidal tumors. By analyzing the ratio of phosphocreatine to phosphodiesters spectral area values, we interpreted qualitatively spectra of intraocular tumors to differentiate malignant tumors from benign lesions. Nevertheless, the main limitation of interpreting the spectra was their contamination by signals from surrounding tissues.     

31P nuclear magnetic resonance and laser spectroscopic analyses of lens transparency during calcium-induced opacification

The authors studied the relationships between lens phosphate metabolites and transparency in (1) normal intact calf lenses, (2) whole-lens homogenates, (3) cortical homogenates, and (4) cortical homogenates that were opacified by the addition of calcium. Transparency was measured with the use of laser spectroscopy. Phosphate metabolites and pH were measured with the use of phosphorus nuclear magnetic resonance (31P NMR) spectroscopy. 31P NMR spectra of fresh whole-lens homogenates and fresh intact lenses were nearly identical. In fresh tissue, adenosine triphosphate (ATP) accounted for 59%, 55%, and 61% of the total phosphorus detected in intact lenses, whole-lens homogenates, and cortical homogenates, respectively. As ATP decreased over time to undetectable levels, no loss of transparency was measured, indicating no relationship between ATP levels and lens opacification. In contrast, the authors found that the loss of transparency in cortical homogenates produced by additions of 5, 10, and 20 mM calcium chloride was associated with increased levels of sugar phosphates and glycerol phosphorylcholine and with decreased levels of inorganic phosphate. Loss of cortical transparency was associated with both increases or decreases in pH from the normal value.     

An electron microscopic and nuclear magnetic resonance spectroscopic evaluation of rabbit corneal epithelial wound healing.

Phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy is a powerful method that can be used to measure levels of phosphorus metabolites in living tissue. To determine whether NMR measurements adequately describe and quantify the wound-healing process in corneal epithelium, 31P NMR spectroscopic and electron microscopic results were correlated. Dutch belted rabbit corneal epithelium was removed with a limbus-to-limbus scrape wound and sampled from 0-30 days postwounding. Four corneas were pooled for each NMR determination of corneal adenosine triphosphate (ATP). A minimum of three animals from each time point were processed for transmission and scanning electron microscopy (EM). Similar to previous studies, migrating cells completely closed the defect by 4 days postwounding. The epithelium continued to increase in thickness and hemidesmosome density was similar qualitatively to that in control animals by 10 days postwounding. The 31P NMR ATP values showed an increase toward control values after an initial 70-75% decrease, returned to within 90-95% of nonwounded values by 10 days, and returned to control values by 30 days. After these experimental values were obtained, the predictive value of the model was tested using platelet-derived growth factor (PDGF)-treated corneas. The PDGF-treated corneas showed NMR and EM results similar to day-10 untreated corneas by 5 days after wounding. These results demonstrated the feasibility of using 31P NMR spectroscopic measurements of ATP levels as a nondestructive technique for quantifying corneal epithelial wound healing.     

Detection and identification of endogenous small molecules in ocular tissues by proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance (nmr) spectra of rabbit ocular tissue homogenates (corneal epithelium, conjunctiva and iris ciliary body) and aqueous humor have been recorded for the first time by incorporation of a spin-spin relaxation reagent and use of the CPMG pulse sequence. A number of endogenous species such as lactate, glucose, alanine and valine have been observed in these ocular homogenates and significant differences have been noted in the distribution of these small molecules in the ocular tissues studied. This technique has been used to study the hydrolysis of acetylcholine by ocular esterases in the iris ciliary body.     

Phosphorus nuclear magnetic resonance and ocular metabolism

Phosphorus (31P) nuclear magnetic resonance (NMR) represents a noninvasive technique for the assessment of ocular metabolism. The measurement of a spectrum of phosphorus-containing metabolites (e.g., phosphorylated sugars and ATP), including a number of heretofore uncharacterized metabolites, can be made with a single analysis. In addition to quantitating phosphatic metabolites, 31P NMR can be employed to monitor (1) the rate of metabolic change in a specific biochemical reaction via T1 and T2 relaxation times, and (2) the rate of change in the concentration of a particular metabolite. Several calculations indicating tissue energy status (health) can be made using quantitative spectroscopic information including: the phosphorylation potential, the energy charge of the adenylate system, and the 31P spectral modulus. Tissue pH can be determined as a function of shift in 31P NMR signals. 31P NMR techniques have both research and diagnostic applications in ophthalmology since potentially it provides a noninvasive method to analyze ocular tissues metabolically and detect subtle biochemical changes that precede overt manifestation of disease states. Such detection may allow for early and more effective therapeutic intervention of disease. Furthermore, the noninvasive quality of NMR spectroscopy will permit continual evaluation of therapy.     

Effects of moist-chamber and McCarey-Kaufman medium storage on the metabolic status of the cornea: a 31P-magnetic resonance analysis

The rate of change in concentration of corneal phosphatic metabolites of cat corneas stored in moist chamber and McCarey-Kaufman (M-K) medium was determined in order to provide a basis for prediction of the corneal metabolic status at a given storage time. Perchloric acid corneal extracts were examined by phosphorus-31 magnetic resonance after storage at 4 degrees C of whole globes under moist-chamber conditions up to 48 h and of excised corneas in M-K medium up to 168 h. A significant decline in the corneal concentrations of ATP and a significant increase in inorganic phosphate occurred for both storage methods; however, depending on the metabolite, the rate of decline or increase was significantly greater for the moist-chamber-stored corneas. The phosphorylated sugars significantly increased and the glycerophosphodiesters significantly decreased in the moist-chamber-stored corneas, whereas both metabolites remained unchanged in the M-K-medium-stored corneas. There was no significant change in the dinucleotides and nucleoside diphospho-sugars during the time course for both storage methods. A threefold greater rate of decline was noted in the tissue energy modulus for the moist-chamber-stored corneas than for the M-K-medium-stored corneas (-0.0465 vs. -0.0121 modulus values/h). M-K medium was significantly more effective in the maintenance of high-energy phosphatic metabolites. The mathematical model for these rate determinations provides a basis for prediction of the corneal metabolic status at a given time in moist-chamber or M-K medium storage.