Analysis of acidic metabolites of biogenic amines in bovine retina and vitreous and aqueous humour by gas chromatography-negative ion chemical ionisation mass spectrometry.

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.

Precision of estimating the time since death by vitreous potassium--comparison of two different equations.

The precision of death time estimation by vitreous potassium using two different formulas [1-3] is compared on an independent random sample of 100 cases. The very flat slope in Sturner's equation is the reason for a systematic overestimation of the time since death with much wider 95%-limits of confidence compared to the results using an own equation with a steeper slope of vitreous potassium.

Basic fibroblast growth factor levels in the vitreous of patients with proliferative diabetic retinopathy.

A two-site enzyme-linked immunosorbent assay was used to quantitate levels of basic fibroblast growth factor in the vitreous from 36 patients undergoing vitrectomy for a variety of retinal conditions, including proliferative diabetic retinopathy, macular pucker, and retinal detachment with and without proliferative vitreoretinopathy. Basic fibroblast growth factor levels ranged from undetectable to 52 ng/mL. In patients with proliferative diabetic retinopathy, basic fibroblast growth factor levels were greater than or equal to 30 ng/mL in 8 of 17 specimens. Of the 8 patients with elevated basic fibroblast growth factor levels, 6 had evidence of active proliferative disease (ie, neovascularization of the disc or iris), whereas in the patients who had undetectable levels only 2 of 9 had evidence of neovascularization of disc and none had neovascularization of the iris. In the rhegmatogenous retinal detachment group, 2 of 10 eyes had elevated basic fibroblast growth factor levels, while none in the macular pucker group had elevated levels. Our study documents increased levels of basic fibroblast growth factor in vitreous specimens from patients with proliferative diabetic retinopathy, particularly those with active proliferative retinopathy. The role of basic fibroblast growth factor in the pathogenesis of various retinal disease entities is discussed.

The origin of the intrinsic glycoproteins of the rabbit vitreous body: an immunohistochemical and autoradiographic study.

A cartilage matrix glycoprotein (CMGP), previously identified in human and bovine vitreous, now has been found in the vitreous body of rabbits aged 1-22 months by immunohistochemical techniques. Epithelial cells of the inner layer of the ciliary epithelium contain material that has immunologic cross-reactivity with a specific antibody to CMGP. These cells also secrete glycoproteins, as determined by autoradiography after intravitreal injection of [3H]fucose. Approximately 14 bands, representing intrinsic glycoproteins containing fucose residues, can be identified in fluorograms of SDS-polyacrylamide gels of vitreous bodies from 6- and 22-month-old rabbits. Fluorograms of gels of samples of vitreous and ciliary bodies from several time points after intravitreal injection of [3H]fucose reveal at least seven comigrating protein bands and also demonstrate turnover of the labeled ciliary body glycoproteins. These results suggest that the inner layer of the ciliary epithelium is the source of the glycoproteins of the vitreous body and that these glycoproteins undergo turnover, probably throughout the entire life of the animals.

Occurrence in chick embryo vitreous humor of a type IX collagen proteoglycan with an extraordinarily large chondroitin sulfate chain and short alpha 1 polypeptide.

We have prepared a high buoyant density proteoglycan fraction from the vitreous humor of 13-day-old chick embryos. Using immunoblot analysis coupled with chondroitinase digestion, we demonstrate that the purified preparation is composed predominantly of type IX collagen-like chondroitin sulfate proteoglycan with an alpha 1(IX) chain Mr approximately 23,000 shorter than the known alpha 1 in cartilage type IX. Also different from cartilage type IX is the size of the chondroitin sulfate chain attached to the alpha 2(IX) polypeptide; its Mr is approximately 350,000 indicating that it is approximately 10 times larger in vitreous humor than in cartilage. Examination of vitreous bodies at different developmental stages indicates that a transition occurs in the size of alpha 1(IX) in a well defined temporal pattern; at about stage 31, a cartilage-type alpha 1(IX) of Mr 84,000 is the predominant species, whereas at stage 36 and thereafter, a Mr 61,000 species appears with a concomitant disappearance of the Mr 84,000 species. Immunostaining for type IX collagen followed by electron microscopic observation of 13-day-old chick embryo vitreous humor reveals a regular D-periodic arrangement of vitreous type IX collagen proteoglycan along thin fibrils. It seems possible that the chondroitin sulfate chains of extraordinarily high viscosity and high molecular weight may extend away from the fibrils, thus contributing to structural as well as functional properties of this unique matrix.

Melatonin concentrations in the sudden infant death syndrome.

To examine a possible relationship between pineal function and the sudden infant death syndrome (SIDS), samples of whole blood, ventricular cerebrospinal fluid (CSF) and/or vitreous humor (VH) were obtained at autopsy from 68 infants (45 male, 23 female) whose deaths were attributed to either SIDS (n = 32, 0.5-5.0 months of age; mean +/- S.E.M., 2.6 +/- 0.2 months) or other causes (non-SIDS, n = 36, 0.3-8.0 months of age 4.3 +/- 0.3 months). The melatonin concentrations were measured by radioimmunoassay. A significant correlation was observed for melatonin levels in different body fluids from the same individual. After adjusting for age differences, CSF melatonin levels were significantly lower among the SIDS infants (91 +/- 29 pmol/l; n = 32) than among those dying of other causes (180 +/- 27; n = 35, P less than 0.05). A similar, but non-significant trend was also noted in blood (97 +/- 23, n = 30 vs. 144 +/- 22 pmol/l, n = 33) and vitreous humor (68 +/- 21, n = 10 vs. 81 +/- 17 pmol/l, n = 15). These differences do not appear to be explainable in terms of the interval between death and autopsy, gender, premortem infection or therapeutic measures instituted prior to death. Diminished melatonin production may be characteristic of SIDS and could represent an impairment in the maturation of physiologic circadian organization.

Diagnosis of nitrate toxicosis in cattle, using biological fluids and a rapid ion chromatographic method.

An ion chromatographic method was used to simultaneously determine nitrate and nitrite ions in biological samples. Ultrafiltration was used to produce a protein-free filtrate. Chloride interferences were eliminated by precipitation as the silver salt. Detection limits and average recoveries were 0.5 mg/L and 102% for nitrate and 0.2 mg/L and 78% for nitrite, respectively. Nitrate concentration was 2.1 +/- 1.8 mg/L and 4.9 +/- 0.8 mg/L in serum and ocular fluid of healthy cattle, respectively; nitrite was not detected. A severe case of nitrate poisoning in cattle was described and used to study the concentrations of nitrate and nitrite in samples obtained under natural conditions. Nitrate concentration of acutely poisoned cattle was 35% lower in ocular fluid at 158.1 +/- 51.4 mg/L, than in serum at 256.3 +/- 113.4 mg/L. Nitrite was not detected, because of the long processing time (greater than 3 hours) required for samples obtained in the field. A gradual decrease in ocular fluid nitrate of 29.4% at 24 hours, 25.9% at 36 hours, 51.6% at 48 hours, and 73.2% at 60 hours was observed; however, concentrations remained diagnostically significant (73.2 mg/L) 60 hours after death. Twenty-four hours after poisoning, the serum nitrate concentration of severely ill (52.7 +/- 51.9 mg/L) and moderately affected (12.4 +/- 5.7 mg/L) cattle that survived was indicative of the severity of clinical signs previously observed. Nitrate in serum and ocular fluid was stable in samples stored for 24 hours at 23 C, 1 week at 4 C, and 1 month at -20 C.

Isotachophoretic determination of lactate ions in vitreous humour samples and its use in forensic toxicology.

Isotachophoresis has been successfully used to determine the lactate ion concentrations in 40 vitreous humour samples taken from subjects representative of those normally encountered by the forensic pathologist. The method is described, and the use of the analysis in forensic toxicology is discussed.

[Quantitative determination of 5 vitreal proteins in the normal vitreous body and proliferative retinal diseases]. / Quantitative Bestimmung von fünf Glaskörperproteinen im normalen Glaskörper und bei proliferativen Netzhauterkrankungen.

While progress in vitreoretinal surgery has been very rapid in recent years, our understanding of the biochemistry of normal and pathological vitreous is still limited. Therefore, we developed non-competitive ELISA techniques for the quantification of five vitreal proteins which, together with vitreal collagen (300 mg/l), account for more than 70% of the total vitreal protein. Physiological levels of the individual proteins were determined as follows: albumin 293 +/- 18 mg/l, IgG 34 +/- 3 mg/l, transferrin 74 +/- 7 mg/l, alpha 1-antitrypsin 14 +/- 3 mg/l, alpha 1-acid glycoprotein 4 +/- 0.7 mg/l. Mean values for these proteins were also obtained in vitreous aspirates from patients with traumatic proliferative vitreoretinopathy (PVR) (n = 10), idiopathic PVR (n = 10) and proliferative diabetic retinopathy (PDR) (n = 15). Significant differences were found for total vitreal protein and alpha 1-antitrypsin between the control groups and the three vitreoretinal disorders, between the PDR and control group for transferrin, and between both types of PVR and controls for alpha 1-acid glycoprotein. Given the high plasma levels of the individual proteins quantified in this study and the uniform rise in total vitreal protein, a disturbancee of blood-retinal and blood-vitreal barriers seems to be an essential features of proliferative intraocular disorders. No disease-specific change in the protein pattern could be detected for the three disorders examined.

Alcohol screening with the Alcoscan test strip in forensic praxis.

The Alcoscan test strip was applied as an assay for the screening of alcohol in vitreous humor and urine samples in autopsy cases and in saliva from drunken drivers. The method gives instant and reliable semi-quantitative information on the presence of alcohol and is valuable when considering the necessity of chemical sampling especially during autopsy.

An example of cocaine tolerance in a gunshot wound fatality.

A 29-year-old male received a fatal gunshot wound to the head during an altercation. Routine toxicological followup on the victim revealed a concentration of 30 mg/L of cocaine in the blood. The case history presented suggests that the concentration achieved by this individual was the result of a physiological tolerance developed from years of consistent recreational use and not by an intentional or accidental overdose.

Renin in the bovine eye.

Recently, high levels of prorenin were found in human vitreous and subretinal fluid. In this study we attempted to identify and quantitate renin and prorenin in the bovine eye. Both these substances are present in the bovine eye in concentrations that cannot be explained by plasma contamination. Concentrations of total renin, i.e. renin plus prorenin, are highest in the posterior uveal tract [15.4 ng angiotensin (Ang) l/g per h]; in the anterior uveal tract the total renin concentration was 10.1, in plasma 6.3, in vitreous fluid 5.7 and in the retina 5.1 ng Ang l/g per h. Vitreous fluid contains mainly prorenin (99%), whereas the retina, and the anterior and posterior uveal tract contain less prorenin (respectively, 78, 47 and 32%). The absence of renin in vitreous fluid is consistent with the general finding that extrarenal renin synthesis is often associated with the release of mainly or exclusively prorenin into the extracellular fluid. Synthesis of renin and prorenin may take place in the eye.

A SepPak HPLC method for tricyclic antidepressant drugs in human vitreous humor.

Death from tricyclic antidepressant overdose has become an all-too-common occurrence. Several factors, including postmortem concentration changes, can render blood and tissue samples useless for the determination of antidepressant drug concentrations. We present here an efficient method of solid-phase extraction for these drugs from vitreous humor and a reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous quantitation of amitriptyline, doxepin, and imipramine and their desmethylated metabolites.

A comparative study of the microbiological contamination of postmortem blood and vitreous humour samples taken for ethanol determination.

Postmortem blood and vitreous humour samples were taken from each of 51 subjects. None of the vitreous humour samples contained large numbers of bacteria or fungi, whereas many micro-organisms were detected in 32 of the blood samples. The results of the microbiological examinations provided useful information for the interpretation of some ethanol levels that might otherwise have been misleading.

Vitreous potassium as a measure of the postmortem interval: an historical review and critical evaluation.

This is an historical review of the articles published in English on the use of vitreous potassium to determine the PMI. External factors which influence the validity of the test are sampling techniques, analytical instrumentation and environmental temperature during the PMI. Internal factors that are recognized at the present time which influence vitreous potassium are the age of the individual, the duration of the terminal episode and the presence or absence of nitrogen retention.

[Breakdown of the blood-retino-vitreal barrier after photocoagulation]. / Rupture de la barrière hémato-rétino-vitréenne après photocoagulation.

The degree of breakdown of the blood-retino-vitreal barrier after Argon laser photocoagulation, and the possibility of preventing it by topical or oral administration of indomethacin, have been investigated in a study involving 58 eyes of 29 rabbits. Protein levels in the vitreous, as determined by densitometry, were four times higher in photocoagulated than in non-photocoagulated eyes (p less than 0.005). Neither topical nor oral administration of indomethacin was successful in preventing post-photocoagulation breakdown of the blood-retino-vitreal barrier at the dosages employed.

Transscleral and transcorneal iontophoresis of ketoconazole in the rabbit eye.

The authors assessed the efficacy of transscleral and transcorneal iontophoresis of ketoconazole as a method of drug delivery to the aqueous humor, vitreous, and cornea of the rabbit eye. Transscleral iontophoresis (4-6 mAmps for 15 minutes) achieved peak ketoconazole concentrations in the aqueous 1 hour after treatment (10.2 micrograms/ml) and remained at fungicidal therapeutic concentrations for 8 hours; in the vitreous, a peak concentration of 0.1 microgram/ml occurred between 1 and 2 hours posttreatment. Transcorneal iontophoresis (1.5 mAmps for 15 minutes) achieved peak corneal concentration of 27.6 micrograms/ml and peak aqueous concentrations of 1.4 micrograms/ml, both 1 hour after iontophoresis. Fungicidal therapeutic drug concentrations were sustained for 2 hours both in the cornea and in the aqueous. These concentrations were compared with those obtained after subconjunctival injection (peak values): 0.8 microgram/ml in aqueous, 5.9 micrograms/ml in cornea, and 0.7 microgram/ml in vitreous, all within 1 hour of injection. Aqueous and corneal concentrations were significantly higher after transscleral and transcorneal iontophoresis than subconjunctival injection (P less than 0.05). Iontophoresis is proposed as an effective means of delivering high concentrations of ketoconazole to the anterior segment of the eye.