Saliva Malondialdehyde Concentration of Dogs With and Without Periodontal Disease.

The study investigated whether malondialdehyde (MDA), a biomarker for oxidative stress, can be used as a viable parameter in dog saliva for the diagnosis or early detection of periodontal disease (PD). Saliva MDA concentrations were measured preoperatively in dogs diagnosed with PD during dental prophylaxis and compared with those of dentally healthy dogs. 35 dogs were included in the study. The average MDA concentration was 270 ng/ml (range 27-633) in the dogs without PD (n = 10) and 183 (36-833) ng/ml (ng/ml) in the dogs with PD (n = 25). The maximum MDA concentration in the study group (PD ≥1) was 833 ng/ml, which was significantly higher than in the study group (PD = 0) (p<0.05). The study showed that salivary MDA concentrations could not distinguish between healthy dogs and those with PD.

Polarization-modulated second harmonic generation imaging: method for quantitative assessment of disorganization in anulus.

An experimental method for quantifying disorder within the anulus fibrosus is described based on polarization-modulated second harmonic generation imaging (PM-SHG-I). This method is demonstrated by imaging the anular lamellar architecture of a mouse model of compressive loading. Results were consistent with those obtained in an earlier study where organization was quantified directed secants image analysis on photomicrographs. In this study the orientation within individual lamellia is quantified by average orientation of the collagen molecules within a defined volume of a single lamellar as measured by the PM-SHG-I. Lamellar boundaries can be identified through the SHG intensity images, and confirmed through co-registration with photomicrographs of the same region. The orientation within the lamellar is quantified by the polarization angle of the maximum second harmonic intensity. PM-SHG-I offers several advantages as compared with the method of directed secants: first, it is nondestructive, allowing repeated measurements of the same tissue; second, images are captured on the order of seconds and capable of obtaining information up to a depth of 200-300 microns, thus allowing for real-time assessment of load damage; third, organization is measured at a much higher resolution, as it is based on disorder within the molecular arrays of a single lamella.

Chronic estradiol treatment attenuates stiffening, glycoxidation, and permeability in rat carotid arteries.

Aging-related changes in vascular stiffening and permeability are associated with cardiovascular disease. We examined the interaction of estradiol on the aging process in vascular tissue from rats by assessing the changes in endothelial layer permeability, arterial compliance, and glycoxidative damage levels. We isolated carotid arteries from ovariectomized (OVX) rats that underwent 1 yr of estrogen treatment with subcutaneous pellets and a subsequent 1 mo of cessation of treatment. Endothelial layer permeability and arterial compliance were determined using quantitative fluorescence microscopy. Endothelial layer permeability was reduced with estradiol treatment (estrogen groups, 2.58 +/- 0.21 ng dextran x min(-1) x cm(-2) vs. nonestrogen groups, 4.01 +/- 0.30 ng dextran x min(-1) x cm(-2); P < 0.05). Additionally, arteries from animals treated with estradiol had an increased compliance index (estrogen groups, 82.9 +/- 3.8 mm2. Torr vs. nonestrogen groups, 69.3 +/- 3.2 mm2. Torr; P < 0.05). Estradiol treatment also reduced levels of pentosidine, which is a specific marker of glycoxidative damage (estrogen groups, 0.11 +/- 0.03 pmol pentosidine/nmol collagen vs. nonestrogen groups, 0.20 +/- 0.03 pmol pentosidine/nmol collagen; P < 0.05). These results indicate that estradiol has multiple chronic vasculoprotective effects on the artery wall to maintain normal vascular wall function.

Vascular dysfunction induced by AGE is mediated by VEGF via mechanisms involving reactive oxygen species, guanylate cyclase, and protein kinase C.

OBJECTIVE: These experiments were designed to elucidate mechanisms mediating vascular dysfunction induced by advanced glycation end products (AGEs). METHODS: Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 week later, granulation tissue that formed in the bottom of the chamber was exposed twice daily for 7 days to glycated rat serum albumin in the presence and absence of inhibitors of reactive oxygen intermediates, nitric oxide synthase and guanylate cyclase, protein kinase C (PKC), and a neutralizing vascular endothelial growth factor (VEGF) antibody. Vascular (125)I-albumin clearance and blood flow were quantified by use of a double isotope-dilution technique and radiolabeled microspheres, respectively. RESULTS: Albumin permeation and blood flow were increased dose-dependently to a maximum of 2 to 3 times controls by increasing the extent of glucose modification, the concentration, or the duration of exposure to glycated albumin. These increases were significantly attenuated by probucol and superoxide dismutase; N(G)-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase inhibitor; LY83583, a guanylate cyclase inhibitor; and LY333531, a beta-isoform-selective protein kinase C inhibitor. A neutralizing VEGF monoclonal antibody also markedly attenuated the permeability and blood flow increases induced by glycated albumin. CONCLUSIONS: These observations indicate potentially important roles for oxygen free-radicals and nitric oxide in mediating permeability and blood flow changes induced by glycated proteins via mechanisms involving increased protein kinase C activity and VEGF production. Striking similarities in the mechanism by which hyperglycemia and glycated proteins induce vascular dysfunction suggest that a common pathway mediates effects of these different metabolic imbalances on vascular dysfunction.

Collagen structure and nonlinear susceptibility: effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity.

BACKGROUND AND OBJECTIVE: Helical macromolecules such as collagen and DNA are characterized by nonlinear optical properties, including nonlinear susceptibility. Because collagen is the predominant component of most biological tissues, as well as the major source of second harmonic generation (SHG), it is reasonable to assume that changes in harmonic signal can be attributed to structural changes in collagen. The purpose of this study is to determine whether various modifications of collagen structure affect second harmonic intensity. STUDY DESIGN/MATERIALS AND METHODS: SHG was measured in tissues from cows, humans, and chickens. The effects of beam polarization, thermal denaturation, glyco-oxidative damage, and enzymatic cleavage of tissues on second harmonic intensity was studied. RESULTS: The second harmonic intensity differed considerably among different tissues, as did the effect of the incident beam polarization. In structurally modified collagen, SHG was significantly degraded from SHG in intact collagen. CONCLUSION: These structural modifications are representative of changes that occur in pathophysiologic conditions such as thermal injury, diabetes, tumor invasion, and abnormal wound healing. The ability to assess these changes rapidly and noninvasively has considerable clinical applicability. SHG analysis might provide a unique tool for monitoring these structural changes of collagen.

Analysis of chromate-induced DNA-protein crosslinks with the comet assay.

Modifications of the comet assay have been introduced to measure crosslinks by determining the reduction of induced DNA migration. Our previous results indicated that the modified protocol of the alkaline comet assay is a sensitive tool for the detection of formaldehyde-induced DNA-protein crosslinks. But results for mitomycin C and cisplatin suggested that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. We now used the comet assay to investigate in V79 cells the effect of potassium chromate (K(2)CrO(4)), another DNA-protein crosslinker, to see whether the results obtained for formaldehyde can be generalized. However, chromate did not reduce spontaneous or radiation-induced DNA migration in the alkaline (pH 13) comet assay but led to a small but significant induction of DNA migration. A crosslinking effect of chromate could also not be detected with the alkaline comet assay after postincubation of cells in normal medium after chromate treatment to enable repair of other (migration-inducing) lesions that might mask the crosslinking effect. Exposure of slides to proteinase K further increased DNA migration of chromate-treated cells, thus indicating the presence of DNA-protein crosslinks. In contrast to the alkaline comet assay, a "neutral" version at pH 9 was suited to demonstrate reduced induction of DNA migration after gamma-irradiation of chromate-treated cells. The crosslinking effect was seen immediately at the end of the chromate treatment as well as after a 3h postincubation period. Using the "neutral" protocol in combination with proteinase K, we were able to demonstrate the presence of DNA-protein crosslinks as the probable cause for the migration-reducing effect. Further investigations will have to show whether this protocol can be recommended as a universal approach for the detection of DNA-protein crosslinks and also of DNA-DNA crosslinks with the comet assay.

17 beta-estradiol reduces glycoxidative damage in the artery wall.

Glycoxidative damage in the vasculature has been linked to atherosclerotic cardiovascular disease. Estrogens protect against the development and progression of atherosclerosis. Because estrogens are potent antioxidants that also effect glucose metabolism, part of their protection against atherosclerosis could be through attenuation of glycoxidative damage in the vascular wall. In this study, we tested the hypothesis that chronic estradiol administration is associated with decreased levels of glycoxidative damage in arterial walls. We harvested and examined iliac arteries from ovariectomized, 8-month-old rats that had been implanted for 6 months with 1 of the following subcutaneous hormone pellets: low estradiol (2.5 mg estradiol), high estradiol (25 mg estradiol), P4 (200 mg progesterone), low estradiol and P4, placebo (no hormone), or control (no implant). Using pentosidine as a biomarker of glycoxidative damage, we found that all vessels from rats receiving estradiol (low estradiol, high estradiol, and low estradiol+P4) exhibited a 50% reduction in glycoxidative damage compared with P4, placebo, and control vessels (P<0.05). Consistent with this finding, we observed that estradiol-treated rats had a 30% decrease in tissue levels of hydroperoxides, a marker of oxidative stress. Finally, estradiol-treated rats had a small, but significant, decrease in plasma glucose levels (P<0.01). In summary, we report the novel finding that chronic estrogen administration is associated with significant decreases in glycoxidative damage and oxidative stress in the arterial wall. It seems likely that these actions may constitute a mechanism by which estrogen attenuates the progression of atherosclerosis.

Rhinoresistometry versus rhinomanometry--an evaluation.

Allergic nasal hyperreactivity is a common problem and many patients suffer from daily symptoms. Rhinomanometry is so far the only well established clinical method for objective assessment of nasal patency, although several expressions of nasal patency have been reported. Universal standardisation was achieved in 1983 in Brussels by Clement et al. [1], but many specialists are looking for a system giving more information on the functional aspects of the nose. A new development arising from active anterior rhinomanometry is rhinoresistometry. We tested this equipment, which has been introduced with new software for calculation and graphic presentation. 24 adult volunteers with proven allergy to grass pollen were examined immediately after long-term challenge in the Vienna Challenge Chamber [3] and 15 minutes after decongestion by application of 5% ephedrine solution. The similarity and differences between rhinomanometry and rhinoresistometry, as well as the value of the additional parameters are pointed out. Our data indicate that rhinoresistometry is a rapid, reproducible and non-invasive technique, which gives extended information in comparison to classic rhinomanometry. The results correlate very well with the findings obtained by the standard method. This pilot study demonstrates the benefit of the new parameters.

The advanced glycation endproduct pentosidine induces the expression of PDGF-B in human retinal pigment epithelial cells.

Advanced glycation endproducts have been implicated in a number of diabetic and aging changes. Some of these effects occur in part through induction of cytokines such as platelet-derived growth factor (PDGF), which is expressed by the retinal pigment epithelium (RPE). In this study, cultures of RPE were evaluated for PDGF expression after treatment with pentosidine, a well characterized advanced glycation endproduct. Northern analysis provided evidence for the increased expression of a 3.7 kb PDGF-B transcript over unstimulated controls in the established ARPE-19 cell line. Western analysis demonstrated increased PDGF-BB protein in conditioned medium compared to controls of ARPE-19 cells. In addition, two different early passage cultures of RPE showed increased PDGF-BB protein after pentosidine treatment compared to unstimulated controls. The enhanced production of PDGF-BB could play a role in the maintenance of the RPE-Bruch's membrane complex and influence changes associated with diabetes and aging.

Effects of aspirin or basic amino acids on collagen cross-links and complications in NIDDM.

OBJECTIVE: To determine if long-term therapy with aspirin or basic amino acids for subjects with NIDDM reduces the severity of clinical complications and/or reduces tissue levels of markers of glycooxidative damage. RESEARCH DESIGN AND METHODS: Subjects with NIDDM were administered either aspirin (100 mg/day) or a combination of basic amino acids consisting of L-arginine (2 g/day) plus L-lysine (0.5 g/day) for 1 year. The study was double-blind and placebo-controlled. The presence and severity of retinopathy, nephropathy, and neuropathy were assessed in all subjects at 4-month intervals, as were serum blood glucose, glycohemoglobin levels, and presence of albuminuria. Collagen cross-linking and collagen glycation were measured in skin collagen obtained by biopsy at the beginning and the end of the study. Skin biopsies were also obtained from age-matched control subjects. RESULTS: Skin samples obtained from NIDDM subjects at the beginning of the study had significantly increased levels of glucitolyllysine, pentosidine, and hydroxypyridinium, as compared with age-matched control subjects. Pentosidine levels were significantly correlated with severity of retinopathy and neuropathy, but not nephropathy. Subjects receiving aspirin, but not amino acids or placebo, had significantly decreased levels of skin pentosidine after 1 year of therapy. CONCLUSIONS: It is concluded that 1) low-dose aspirin may reduce glycooxidative damage in people with NIDDM, and 2) treatment may need to continue for more than 1 year before clinical status improves.

[Reliability of expert assessment in disability evaluation within the scope of disability insurance]. / Reliabilitätsstudie zum Begutachtungsverfahren der Pflegebedürftigkeit im Rahmen der Pflegeversicherung.

With the introduction of the law on the statutory nursing care insurance in Germany ("Pflegeversicherung") in 1995, new benefits are provided for the most severely disabled people. Provision of benefits is contingent on a standardised examination performed by the medical service of the German statutory health insurance system (MDK). Results of the first study on the inter-rater reliability of this standardised examination are presented in this paper. The study population consisted of 218 elderly people (age range 60-99) living in 4 nursing homes in Munich. They were assessed by 2 senior medical students and 2 nurses using the standardised questionnaire of the MDK. Inter-rater reliability was assessed by means of kappa coefficients. Reliability was assessed with regard to the summary judgement of disability and the rating of single items (with regard to limitations in activities of daily living, mental status or the prognosis). Inter rater reliability was higher for the overall assessment of disability (kappa = 0.71 between medical students, kappa = 0.66 between nurses) than for most of the single items of the questionnaire such as "need of assistance with dressing" (kappa = 0.57 between medical students, kappa = 0.66 between nurses) or "ability to move" (kappa = 0.58 between medical students, kappa = 0.67 between nurses). Kappa-coefficients were particularly low for variables concerning prognosis of participants and for the mental status item "self-disorientation" (kappa = 0.19 between medical students, kappa = 0.23 between nurses). These patterns were consistently observed for the 2 rater groups (student-student, nurse-nurse). Between the 2 rater groups there was no substantial difference in reliability. However, there was a trend for higher agreement within the group of nurses. Although the overall reliability of the assessment of disability is relatively high in comparison to many other diagnostic procedures (such as x-ray readings), efforts should be made towards further improvement of this standardised examination.

Effect of maturation and aging on the histomorphometric and biochemical characteristics of equine superficial digital flexor tendon.

OBJECTIVE: To assess tendon morphology and non-reducible crosslink concentration, and associations of these findings with horse age and previously reported mechanical and ultrasonographic findings. SAMPLE POPULATION: Superficial digital flexor tendon samples were obtained from 23 horses aged 2 to 23 years. The tendons had undergone ultrasonography and were submitted to biomechanical testing in the physiologic range prior to sample acquisition. PROCEDURE: Samples were sectioned in a transverse plane; then dorsal, palmar, central, lateral, and medial regions were evaluated for fascicle cross-sectional area (CSA), septal width, and vessel density (the product of vessel numbers and vessel CSA per field). Contiguous samples were analyzed for collagen crosslinking. RESULTS: Central fascicles were significantly larger than fascicles in other tendon regions. Fascicle CSA decreased significantly with increasing age. Because total tendon CSA is unrelated to increasing age, fascicle numbers appeared to increase with increasing age. Regional or age effects on septal width were not found. There was no age or regional effect on vessel numbers, density, or fractional area. Fascicle CSA was positively correlated with total tendon CSA; fascicle CSA was negatively correlated with elastic modulus. Hydroxypiridinium concentration tended to increase with increasing horse age; this effect was associated with a positive correlation between hydroxypiridinium values and elastic modulus. CONCLUSIONS: Equine superficial digital flexor tendon undergoes an increase in structural organization and an increase in nonreducible crosslinks with maturation and aging. These changes are associated with an increase in elastic modulus.

Discordant effects of guanidines on renal structure and function and on regional vascular dysfunction and collagen changes in diabetic rats.

We examined the effects of aminoguanidine and methylguanidine on vascular dysfunction, glomerular structural changes, and indexes of early and late nonenzymatic glycation in 7-month streptozotocin-induced diabetic rats. Kidney weight, glomerular volume, fractional mesangial volume, glomerular capillary basement membrane width, and urinary albumin excretion were increased in diabetic rats. Diabetes also 1) increased vascular albumin permeation twofold in retina, sciatic nerve, aorta, skin, and kidney; 2) decreased renal collagenase-soluble collagen; 3) increased collagen-associated fluorescence in kidney and skin but not in aorta; and 4) increased glycated hemoglobin levels and aortic pentosidine levels. Aminoguanidine reduced albuminuria by 70% after 4 months, and both guanidines 1) normalized aortic pentosidine levels and renal collagenase-soluble collagen, 2) had no effect on glycated hemoglobin levels or collagen-associated fluorescence (in aorta, kidney, or skin), and 3) had little or no effect on regional albumin permeation. These discordant effects of aminoguanidine on diabetes-induced vascular changes versus parameters of nonenzymatic glycation are consistent with a multifactorial pathogenesis of diabetic complications, including roles for metabolic imbalances independent of nonenzymatic glycation. To the extent that glomerular matrix accumulation and increased regional albumin permeation in chronically diabetic rats are sequelae of nonenzymatic glycation, these findings point to an important role for early glycation reactions and products.

Effects of elevated circulating IGF-1 on the extracellular matrix in "high-growth" C57BL/6J mice.

Collagen biosynthesis was analyzed in C57BL/6J mice homozygous for the high-growth locus. Plasma levels of insulin-like growth factor-1 (IGF-1) were significantly elevated in high-growth mice at all ages studied (3 wk-6 mo); IGF-binding proteins were also elevated. Skin biopsies were obtained from mice aged 3, 6, and 9 wk under halothane anesthesia. Mice were killed at 6 mo of age. Collagen, expressed per weight of tissue, was significantly increased in all tissues from high-growth mice, as was collagen cross-linking, expressed as moles of cross-link per mole of collagen. Expression of types I and III collagen, lysyl oxidase, and lysyl hydroxylase was increased in all tissues analyzed. There was a preferential increase in type III expression relative to type I expression. Rate and extent of accumulation of collagen in granulation tissue were measured in polyvinyl alcohol sponges implanted subcutaneously; collagen accumulation was significantly greater in the high-growth mice. These results suggest that 1) elevated circulating IGF-1 may increase collagen deposition both in normal tissue as well as in granulation tissue by increasing collagen gene expression, 2) IGF-1 may increase collagen cross-linking by stimulating expression of lysyl oxidase, and 3) the preferential increase in dihydroxylated cross-links observed in high-growth mice may be due to the stimulation of lysyl hydroxylase expression by IGF-1. In summary, elevated levels of IGF-1 appear to affect collagen both quantitatively and qualitatively, primarily through their effects on gene expression of collagen and of those enzymes responsible for posttranslational modifications of collagen.

Lung collagen cross-links in rats with experimentally induced pulmonary fibrosis.

Rats were intratracheally instilled with bleomycin or with silica (quartz) dust to induce lung fibrosis. Several weeks later, purified collagen chains (or collagen digests) were isolated from the lungs of these animals and from age-matched controls instilled intratracheally with saline solution, and the ratios of hydroxylysine to lysine and of the dysfunctional cross-links DHLNL to HLNL were quantified. Collagen from fibrotic lungs had significantly higher ratios of DHLNL:HLNL than did control lungs, 15.5 +/- 4.8 and 17.1 +/- 4.8 vs. 2.3 +/- 0.5 for the silica-instilled and the bleomycin-instilled animals, respectively. The hydroxylysine:lysine ratio was significantly increased for the alpha 1(I) chain, to a value 170% of that of lung collagen from control animals, and for several of its constituent CNBr peptides. Lung tissue was exhaustively digested with collagenase and specific cross-linked peptides were isolated and characterized. The cross-linked alpha 1(I) x alpha 1(I) peptide linked by the residues 87 x 16C, with a ratio of DHLNL:HLNL of 17:1, demonstrated that the increased hydroxylation of the dysfunctional cross-links in fibrotic lung collagen could be accounted for in part by increased hydroxylation of the lysine residue at position 16C of the C-terminal telopeptide of the collagen alpha 1(I) chain. It proved impossible to locate the corresponding N-terminal cross-linked fragment from alpha 1(I) x alpha 1(I) chains, 9N x 930, possibly due to further reactions of this material to form the material referred to as poly(CB6). Isolated poly (CB6) accounted for more than half of the total alpha 1(I)CB6 peptide expected in lung collagen, and had a hydroxylysine:lysine content 2.8 times greater in bleomycin-treated animals than in their age-matched controls. Evidence was also found for a cross-linked alpha 1(III) x alpha 1(I) peptide linking residue 87 from the alpha 1(III) chain with residue 16C from the alpha 1(I) chain; it also had an increased ratio of DHLNL:HLNL. We conclude that the increased hydroxylation of lysine observed in two different animal models of lung fibrosis occurs preferentially at the N- and C-terminal nonhelical extension peptides of the alpha 1(I) collagen chains, and that this apparent specificity of overhydroxylation of fibrotic collagen may have important structural and pathological consequences.

Effect of aminoguanidine on the frequency of neuroaxonal dystrophy in the superior mesenteric sympathetic autonomic ganglia of rats with streptozocin-induced diabetes.

Aminoguanidine, which prevents formation of advanced glycation end products and is a relatively selective potent inhibitor of the inducible (versus constitutive) isoform(s) of nitric oxide synthase, has been reported to ameliorate structural and functional abnormalities in peripheral somatic nerves in rats with streptozocin (STZ)-induced diabetes. In the present studies, the effects of aminoguanidine treatment on ultrastructural changes in the autonomic nervous system of rats with STZ-induced diabetes were examined. The frequency of neuroaxonal dystrophy, the neuropathological hallmark of sympathetic autonomic neuropathy in diabetic rats, increased 9- to 11-fold in the superior mesenteric ganglia of 7- and 10-month STZ-diabetic rats compared with that in age-matched controls. Administration of aminoguanidine continuously from the time of induction of diabetes at a dose equal to or in excess of that providing a salutary effect in the diabetic somatic peripheral nervous system did not alter the severity of diabetes as assessed by plasma glucose level, 24-h urine volume, and levels of glycated hemoglobin. Chronic aminoguanidine therapy did not diminish the frequency or affect the ultrastructural appearance of neuroaxonal dystrophy in diabetic or age-matched control rat sympathetic ganglia after 7 or 10 months of continuous administration. Our findings (under these experimental conditions) do not support a role for aminoguanidine-sensitive processes in the development of sympathetic neuroaxonal dystrophy in diabetic rats. Glycation-linked aminoguanidine-insensitive processes, however, such as the formation of early glucose adducts (Schiff bases and Amadori products) with intracellular and/or extracellular proteins and amine-containing lipids, superoxide anion generation during subsequent autoxidation of these glucose adducts, and non-glycative processes, remain potential pathogenetic mechanisms for diabetic autonomic neuropathy.

Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence.

A fundamental question in the basic biology of aging is whether there is a universal aging process. If indeed such a process exists, one would expect that it develops at a higher rate in short- versus long-lived species. We have quantitated pentosidine, a marker of glycoxidative stress in skin collagen from eight mammalian species as a function of age. A curvilinear increase was modeled for all species, and the rate of increase correlated inversely with maximum life-span. Dietary restriction, a potent intervention associated with increased life-span, markedly inhibited glycoxidation rate in the rodent. On the assumption that collagen turnover rate is primarily influenced by the crosslinking due to glycoxidation, these results suggest that there is a progressive age-related deterioration of the process that controls the collagen glycoxidation rate. Thus, the ability to withstand damage due to glycoxidation and the Maillard reaction may be under genetic control.

Modulation of lysyl oxidase by dietary copper in rats.

Lysyl oxidase levels were estimated in rat tissues using an enzyme-linked immunosorption assay (ELISA) and a functional assay standardized against known amounts of purified lysyl oxidase. High concentrations of lysyl oxidase (> or = 150 micrograms/g of tissue or packed cells) were detected in connective tissues, such as tendon and skin. Values for aorta, kidney, lung and liver ranged from 30 to 150 micrograms/g of tissue; values for skeletal muscle and diaphragm were < 30 micrograms/g tissue. Purified rat skin lysyl oxidase catalyzed the release of 50-100 Bq of tritium per micrograms enzyme in assays that used 3H-elastin-rich substrates. In dense connective tissues, good agreement was obtained for the values from ELISA and those derived from measurements of functional activity in aorta, lung, skin and tendon (r2 > 0.9). When egg white-based experimental diets containing 2 or 10 micrograms/g added copper were fed to weanling rats, values for skin lysyl oxidase functional activity in the group fed 2 micrograms/g added copper were one-third to one-half the values for skin lysyl oxidase functional activity in rats fed 10 micrograms/g copper. This reduction in lysyl oxidase activity, however, had minimal effect on indices of collagen maturation in rat skin, e.g., collagen solubility in neutral salt and dilute acid or the levels of acid stable cross-links. Moreover, copper deficiency did not influence the steady-state levels of lysyl oxidase specific mRNA in rat skin or the apparent amounts of lysyl oxidase in rat skin as determined by ELISA. These observations underscore that the concentration of lysyl oxidase is relatively high in dense corrective tissues, and although decreasing dietary copper influences functional activity, there is little apparent effect on the production of lysyl oxidase protein.