A comparative study of iron-related metabolic parameters in the eye of three animal species

In order to obtain a wider perspective of iron homeostasis in the eye, a comparative study was undertaken of several iron-related parameters (Total Iron, TI; Total Iron Binding Capacity, TIBC; Transferrin, Tf; and saturation of Tf) both in blood serum and in ocular tissues (lens, cornea, iris-ciliary body, retina) and fluids (vitreous body, aqueous humor) of several animal species (pig, cow and rat). The relative degree of oxidative stress of tissues and fluids was evaluated based on the criteria that high values of TI and Tf saturation, and low values of TIBC and Tf, would promote iron-related oxidative stress. The inclusion of both diurnal (pig, cow) and nocturnal (rat) animal species in this comparative work provided the opportunity to explore if iron homeostasis parameters are in some way influenced by the higher oxidative stress level expected in animals with diurnal living habits. This project involved also the design of new and very sensitive methods of analyses, given the fact that in many cases very small amounts of sample (i.e., aqueous humor), and/or low concentration of analytes (i.e., transferrin) are available. All results were expressed as concentration relative to mg protein as determined with the Bradford method (microplate assay). When analyzing TI/TIBC it was possible to define a loosely bound iron pool (LBIP) in ocular tissues that was proportional to the degree of vascularization of the tissues analyzed. The comparison of iron related parameters patterns within the eye and among species allowed us to reach the following conclusions: (1) The aqueous humor and vitreous body of cow and pig exhibited highest concentration of Tf and a very low saturation of Tf, while the lowest value of Tf was detected in all species in the lens; (2) TI showed the tendency to be highest in the vitreous body of cow and pig, and lowest in the lens of all three species. The very low iron concentration in the lens may effectively counteract the...

Characterization of hemoglobin Hotel Dieu in a Puerto Rican adolescent.

PURPOSE: Hemoglobin Hotel Dieu (HbHD) is a high-oxygen affinity variant of HbA never before reported in a Hispanic patient. This Hb variant was first reported in 1981 by Blouquit et al. in a white person with erythrocytosis with a substitution in the beta 99 aspartic acid residue by glycine. METHODS: A 13-year-old Puerto Rican boy had pain in his chest, headaches, easy fatigability, and high Hb (as high as 19.1 g/dl). Protein analysis was performed by cellulose acetate, citrate agar, and isoelectric focusing electrophoresis and high-pressure liquid chromatography (HPLC), polymerase chain reaction (PCR) amplification, and DNA sequencing of the second exon of the beta gene in samples obtained from the mother, father, and the patient, and DNA fingerprinting to determine paternity. RESULTS: The variant found in the patient migrated on cellulose acetate electrophoresis to a cathodic position relative to HbF, and a band cathodal to HbA and close to HbF on isoelectric focusing electrophoresis. The patient showed an abnormal well-resolved peak on HPLC with a retention time slightly shorter than that for HbS. DNA analysis by direct sequencing of the PCR product demonstrated heterozygosity for codon 99 (GAT-->GGT) in the patient but not in either parent. DNA fingerprinting by multiplex PCR amplification of three simple tandem repeat loci showed that the patient shared alleles in all three loci with both parents, ruling out nonpaternity. CONCLUSIONS: The protein and DNA analysis indicate that the erythrocytosis is caused by the presence of HbHD in this Hispanic adolescent.

[Hydrogen peroxide in the aqueous humor: 1992-1997]. / Peróxido de hidrógeno en el humor acuoso: 1992-1997.

Ideas prevailing in 1991 on hydrogen peroxide in the aqueous humor are outlined. They are critically examined under the light of our finding that the method used to establish aqueous humor levels of peroxide generates itself peroxide during the short time span of the analysis. This is due to the fact that the probe used, dichlorophenol indophenol (DCPIP), spontaneously auto-oxidizes in the presence of oxygen. It was concluded then that the level of hydrogen peroxide in the aqueous humor cannot be higher than about 0.3 microM, the detection limit of the DCPIP method. It was also concluded that the statement commonly made in the literature that aqueous humor hydrogen peroxide derives from the oxidation of ascorbate, an abundant component of that fluid, is based solely on the use of the DCPIP method, and so could easily be due to a methodological artifact. The same applies to the statement that the levels of hydrogen peroxide are very high in human senile cataracts. The surprising resistance to accept the results and conclusions of our 1992 publication is documented. Finally, the content is discussed of an oral presentation made at the 1997 ARVO Annual Meeting in which an important portion of our results and conclusions was confirmed, perhaps signaling a shift towards a wider acceptance of our findings.

Increased malondialdehyde in peripheral blood of patients with congestive heart failure.

Malondialdehyde (MDA), a marker of lipid peroxidation, was measured in the plasma of patients with congestive heart failure (CHF) having varying degrees of clinical symptoms and in control subjects. The 53 patients studied were divided in two groups based on their left ventricular ejection fraction (LVEF). Group A consisted of 30 symptomatic patients with chronic CHF (NYHA classes II and III) and LVEF < 40%. Group B consisted of 23 asymptomatic patients with LVEF > 40%. Patients in group A (mean LVEF = 28) has a significantly greater history of myocardial infarction (88% vs 48%; p = 0.002) than those in group B. Group B patients and the controls had similar LVEFs (58.0 vs 62.1; p = 0.14). Neither patients in the CHF group nor group B patients showed correlation between MDA values and LVEF, unless control were included. Mean MDA concentrations in groups A (2.65 +/- 1.03 mumol/L) and B (2.1 +/- 0.7 mumol/L) were significantly higher than those in the control group (1.45 +/- 0.77 mumol/L; p < 0.05), supporting the hypothesis that the CHF state and underlying risk conditions appear to be associate with abnormal oxidative stress. Moreover, a significant correlation (r = 0.74; p = 0.0001) was found in group A patients between the MDA values and the duration in years (chronicity) of the CHF state.

Antibodies to lens crystallins after endocapsular cataract surgery.

Levels of serum antibodies against lens alpha, beta H and beta L crystallins were determined in 15 patients before and after endocapsular cataract extraction. The most abundant antibody at the time of surgery was anti-beta L, followed by anti-beta H and anti-alpha. An increase in the basal levels of these antibodies was seen after surgery in 93 percent of the cases. Relative increase was highest for anti-alpha, followed by anti-beta H and anti-beta L. There were individual variations both in the shape of the time-dependent antibody response after surgery and in the relative intensity of the response to the different antigens. The endocapsular technique therefore leads to the release of crystallins into the anterior chamber in sufficient amounts to provoke in many patients a humoral immune response.

Melanins and lens pigments: a comparative study.

Based on previous findings that lens pigments and melanins share many physicochemical properties, human lens pigments and natural (hair) and synthetic melanins were submitted to oxidation with permanganate under strong acidic conditions. This procedure has been utilized for the characterization of melanins and results in the well defined products, thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), which can be quantitated by high performance liquid chromatography (HPLC). PTCA is regarded as a marker of black eumelanins and was therefore a main component of synthetic DOPA-eumelanin and dark hair. Its identity was established by synthesis from 5-hydroxyindole-2-carboxylic acid. TDCA derives from pheomelanins and was therefore an important component of red hair and synthetic GSH-pheomelanin. TDCA was identified by its retention time relative to PTCA. The analysis of a series of cataract digests of increasing pigmentation (type I < type IV < type V) and a purified fraction of lens pigments (DE52 pigment) revealed the presence in these preparations of both PTCA and TDCA. The concentration of TDCA significantly increased with the degree of pigmentation of the digests and reached a maximum in the DE52 pigment. The TDCA/PTCA ratio was high in the lens preparations and comparable to that given by hair pheomelanin. These findings support that pheomelanin is an integral part of lens pigments. By comparing the yields of TDCA in GSH-pheomelanin and in the purified lens pigment, a 9% contribution of pheomelanin to the lens pigment was estimated.

Aqueous humor hydrogen peroxide analysis with dichlorophenol-indophenol.

Hydrogen peroxide is now reported to be a normal aqueous humor component present, in the low microM concentration range, in the animal species which have been studied. This finding was established with the exclusive use of the dichlorophenol-indophenol method of analysis. In this procedure, aqueous humor is added to a blue, oxidized dichlorophenol-indophenol solution. The 605 nm absorbance of this solution immediately decreases in response to the reducing action of ascorbate present in the sample. The extent of reoxidation of the solution upon the addition of peroxidase, as measured by the increase in its 605 nm absorbance, can be quantitatively related to the concentration of H2O2 in the sample. A close examination of this method revealed that reduced dichlorophenol-indophenol spontaneously reoxidizes at a rate of 0.03 nmol min-1 microM-1, with generation of H2O2. H2O2 generation was unequivocally established by analysis of the temporal dependency of the absorbance increase produced by peroxidase in the absence of added H2O2 and by the sensitivity of this phenomenon to catalase. This spontaneous production of H2O2, on the other hand, cannot be attributed to ascorbate auto-oxidation because added ascorbate quantitatively reacts with dichlorophenol-indophenol, provided that an excess of the latter is maintained. This method then has an enormous potential to overestimate H2O2 in any sample. On the other hand, the response of the assay system to a given level of H2O2 depends on the level of reduction previously produced by ascorbate. This results in an artifactual positive correlation between ascorbate and H2O2 levels in samples containing variable amounts of ascorbate. In spite of these serious limitations the method can still be useful to measure H2O2 if appropriate precautions are taken. When using it for the analysis of rabbit aqueous humor H2O2 without correcting for the H2O2 generated during the assay and ignoring differences in the level of ascorbate in the samples, we obtained an average value of 25.3 microM H2O2, which coincides with that reported in the literature for the rabbit, but is obviously incorrect. When analysing aqueous humor there was the additional variable of the aqueous humor itself inhibiting the rate of dichlorophenol-indophenol auto-oxidation and so the final, corrected figure for H2O2 concentration in the aqueous humor became uncertain, since the auto-oxidation of the substrate could not be properly subtracted.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of nasal instillation of female urine or vaginal exudate on serum cortisol and prolactin levels in isolated and anesthetized male rhesus monkeys (Macaca mulatta).

Two male adult rhesus monkeys were used and caged individually. The room was temperature-controlled having a light-dark period of 12/12 hours. The animals were rapidly immobilized and immediately anesthetized with ketamine i. m. (10 mg/kg of body weight). They were bled four times at 15, 30, 45 and 60 mins after the ketamine injection, twice a week for 6 weeks. When necessary, maintenance doses of ketamine were administered. The serum levels of cortisol and prolactin after nasal instillation of a suspension of vaginal exudate showed lower values than in control conditions (nasal instillation of saline). The control levels of cortisol tended to increase up to 60 mins, whilst in experimental conditions (nasal instillation of female urine or vaginal exudate) did not show such an increase. Cortisol remained similar during the sampling and similar to the 15 mins control levels, but the difference is statistically significant only after instillation of vaginal exudate as compared with control. The levels of prolactin did not show significant variations during sampling either in control or after female urine instillation. However, the instillation of vaginal exudate decreased the prolactin levels at 15 mins which tended to recover the control levels up to 60 mins. These results support the hypothesis, discussed in a previous paper, that some chemical information from females suppresses or mitigates the effect of acute stress resulting from handling the animals before anesthesia.

Effect of nasal instillation of female urine or vaginal exudate on testosterone secretion in isolated and anesthetized male rhesus monkeys (Macaca mulatta).

Two male adult rhesus monkeys were individually placed in cages with a pulling device in order to immobilize the animals for anesthesia. The room was temperature-controlled having a light/dark period of 12/12 hours. The animals were rapidly immobilized and immediately anesthetized with ketamine i. m. (10 mg/kg of body weight). They were bled four times at 15, 30, 45, and 60 mins after the ketamine injection, twice a week during 6 weeks. When necessary, maintenance doses of ketamine were administered. The levels of serum testosterone in experimental conditions (nasal instillation of female urine or a suspension of vaginal exudate) showed significant lower values with respect to those in control conditions (saline instillation). The control levels of testosterone tend to increase up to 60 mins. The testosterone from samples obtained in experimental conditions did not show such an increase, remaining similar during the sampling and similar to the 15 min control levels that could be considered as basal. These results seem to point out some chemical information from females capable of modifying the pattern of secretion of testosterone of the males in the above mentioned experimental conditions.

Computer eyestrain.

Main ocular mechanisms probably underlying eyestrain symptoms in computer users-diminished aqueous tears, extraocular muscle fatigue, color fatigue and decreased amplitude of accommodation induced by monochromatic light--are discussed. With this background, some practical consequences are drawn to avoid or reduce eyestrain.

The endopeptidase-resistant protein fraction from human cataractous lenses.

With aging and cataract formation, modifications in absorption and fluorescence of the human lens proteins are observed. These changes have been investigated by the examination of the endopeptidase-resistant fraction isolated from human cataractous lenses. This fraction is highly enriched in atypical fluorescence and absorption (i.e. not attributable to tryptophan, tyrosine or phenylalanine). It has a molecular weight of approximately 3000, is enriched in acidic amino acids and has only a 280 nm shoulder in its u.v. spectrum. The material does not contain detectable levels of malondialdehyde or N-formylkynurenine. Upon acid hydrolysis the fluorescence and u.v. spectra remain unchanged with only a minor degree of cleavage observed. Structural studies on some of the cleavage products indicated the presence of oxindolyl alanine and kynurenine. These compounds could result from photo-oxidation of tryptophan.

Cerebrospinal fluid ascorbic acid levels in neurological disorders.

The ascorbic acid/dehydroascorbic acid system was analyzed in the cerebrospinal fluid (CSF) of 41 patients with different neurological disorders. The chi-square test of covariance analysis revealed in this sample significant differences in the CSF levels of total ascorbic acid when patients were classified by diagnostic categories. The population analyzed contained a group of 18 patients (back pain/sciatica group) in whom no overt neurological abnormalities were disclosed upon evaluation. Taking the CSF levels of total ascorbic acid and dehydroascorbic acid in these patients as the reference (3.57 +/- 0.87 (SD)/100 ml and 0.53 +/- 0.19 mg/100 ml, respectively), it was found that head-traumatized patients showed a significant reduction in the concentration of total ascorbic acid in the CSF. CSF ascorbic acid levels were also significantly lower in patients with increased intracranial pressure (noninfected hydrocephalus group) and in patients with cerebral tumors. Although the CSF concentration of dehydroascorbic acid did not correspondingly increase over the reference values in these three groups of patients, the tendency existed for dehydroascorbic acid to represent in them a higher percentage of total ascorbic acid. After examining different alternatives, it is concluded that the hypothesis of free radical damage to the central nervous system after certain types of injury (trauma, ischemia, and tumors) may provide a satisfactory explanation of our findings. A rationale for the use of vitamin C in the management of some neurological patients is also derived from this work.

Loss of free amino groups in the water-insoluble fraction of nuclear senile cataracts.

Free amino groups were analyzed in the water-insoluble fraction (WIF) of cataractous human lenses of the nuclear sclerosis, pigmented type. Two modified versions of the trinitrobenzene sulfonic acid (TNBS) procedure for the determination of free amino groups in proteins (AFSA Habeeb, Anal Biochem 14:328-336, 1966) were used for this purpose. The concentration of free amino groups in WIF was found to be inversely related to the dry weight of this fraction. Taking WIF weight as a measure of the severity of nuclear cataract, it can be thus said that a relative loss of free amino groups apparently occurs during the insolubilization process associated to this type of senile cataract. The disappearance rate has been estimated as about 0.9%--NH2 per 1 mg of material added to WIF. After considering other possible alternatives (loss of accessibility of amino groups to TNBS, dilution of amino groups by materials with a low primary amine content) we have interpreted this finding as most likely due to the postsynthetic blockade of lysine epsilon-amino functions in WIF proteins. The potential involvement of lysine residues in Schiff base formation is discussed within the context of current views of cataractogenesis. The reaction of lysine epsilon-NH2 with unidentified carbonyl compounds could represent a partial but quantitatively important mechanism of yellowing in nuclear cataracts.

Detection of bityrosine in cataractous human lens protein.

Bityrosine was isolated from the insoluble protein of human cataractous lenses. Identification was based on correspondence with synthetic bityrosine with respect to chromatography, fluorescence, and ultraviolet and mass spectra. It is suggested that the compound may form cross-links with polypeptide chains in old and cataractous lenses, causing significant alteration in native protein structure.