Glucose-induced microvascular functional changes in nondiabetic rats are stereospecific and are prevented by an aldose reductase inhibitor.

Exposure of skin chamber granulation tissue vessels in nondiabetic rats to 11 or 15 mM D-glucose (but not L-glucose or 3-O-methylglucose) twice daily for 10 d induces vascular functional changes (increased albumin permeation and blood flow) identical to those in animals with mild or severe streptozotocin diabetes, respectively. These vascular changes are strongly linked to increased metabolism of glucose via the sorbitol pathway and are independent of nonenzymatic glycosylation as well as systemic metabolic and hormonal imbalances associated with the diabetic milieu. (J. Clin. Invest. 1990. 85:1167-1172.)

Galactose ingestion increases vascular permeability and collagen solubility in normal male rats.

In view of the similarity of cataracts and neuropathy in galactose-fed and diabetic rats, the present experiments were undertaken to determine whether consumption of galactose-enriched diets (10, 25, or 50% by weight) also increases collagen crosslinking and permeation of vessels by 125I-albumin analogous to that observed in diabetic rats. The observations in these experiments: demonstrate that consumption of galactose-enriched diets for 3 wk selectively increases 125I-albumin permeation of the same vascular beds affected in diabetic rats and by diabetic vascular disease in humans (i.e., the aorta and vessels in the eye, kidney, sciatic nerve, and new tissue formed in the diabetic milieu); demonstrate that the susceptibility of the vasculature to aldose reductase-linked injury (increased permeability) varies greatly in different tissues; indicate that collagen solubility (crosslinking) changes in galactose-fed rats differ sharply from those in diabetic rats; and provide new evidence that consumption of galactose-enriched diets induces a hypogonadal state in male rats.

Capillary basement membranes in diabetes.

In discussions of vascular complications of diabetes the fact that capillary basement membranes are, in general, thickened (CBMT) in poorly controlled diabetics is no longer at issue. However, three important questions concerning the pathophysiologic significance of CBMT remain unanswered: (1) How and why do capillary basement membranes thicken in diabetes? (2) What is the functional significance of capillary basement membrane changes in diabetes? (3) What is the nature of the relationship of CBMT to other forms of diabetic vascular disease; in particular, is CBMT observed in tissues amenable to needle biopsy, i.e., skeletal muscle, useful in identifying individuals at high risk for developing clinically significant retinopathy, nephropathy, or atherosclerotic vascular disease? In this survey, we will consider the nature of capillary basement membrane changes in diabetes and subsequently address the above questions.

Basement-membrane thickening and diabetic microangiopathy.

This report considers the pathophysiologic significance of capillary basement-membrane thickening in diabetic nephropathy and retinopathy and the relationship of capillary basement-membrane thickening to increased susceptibility to infections and to increased vascular permeability in diabetes. The evidence available (1) indicates that basement-membrane thickening affects most if not all capillaries of the diabetic and may contribute to increased susceptibility to infection and (2) suggests that increased capillary permeability in diabetes need not be attributed to basement-membrane changes per se, but rather may be due to changes in the cellular elements of the capillary wall.

Histocompatibility antigens and diabetic retinopathy.

Of 160 patients with onset of diabetes at or after 30 years of age, the 84 with no evidence prevalences of HLA-A1 and B8 when compared with the 76 with retinal complications or with the 282 healthy blood donors. In addition, in 90 patients with onset of diabetes before age 30 years, we could confirm the reported significant increase of HLA-B8 and decrease of B7, but no differences were noted between those juvenile-onset diabetics with and those without retinopathy.

Influence of fixation and morphometric technics on capillary basement-membrane thickening prevalence data in diabetes.

Muscle tissue obtained by needle biopsy from 20 diabetic subjects and from 20 age-matched control subjects was divided into two portions, one of which was fixed and processed by our routine procedure (primary glutaraldehyde fixation followed by osmium fixation and embedment in araldite) and the other was fixed initially in osmium tetroxide and embedded in maraglas, the procedure employed by Siperstein et al. Basement-membrane width of capillaries was measured by the 20-point method of Siperstein et al. and by the two-minimum-point technic developed in our own laboratory. Contrary to the experience of Siperstein et al., the prevalence of basement-membrane thickening in diabetic subjects based on mean width values and/or standard deviations in excess of 95 per cent tolerance intervals was highest (65 per cent) in minimum measurements of glutaraldehyde-fixed tissues and lowest (30 per cent) in osmium-fixed tissues (X2 = 4.9123, p less than 0.05). Internal discrepancies in the data of Siperstein et al. indicate that (1) their basement-membrane-width values derived from multiple measurements from control subjects are anomalous (low) and (2) the very high prevalence of basement-membrane thickening they reported in diabetic and in prediabetic subjects and considered as strong support for their conclusion that basement-membrane disease is independent of and precedes glucose intolerance is suspect.

Albumin permeation of new vessels is increased in diabetic rats.

125I-bovine serum albumin (BSA) permeation of the vasculature of 3-wk-old granulation tissue (induced by subcutaneous implantation of polyester fabric) formed in the diabetic milieu was assessed in female BB/W, spontaneously diabetic rats and in male, Sprague-Dawley rats with streptozocin-induced diabetes as well as in corresponding nondiabetic controls. Albumin permeation of new granulation tissue vessels was markedly increased in both groups of diabetic animals relative to that of nondiabetic controls, while albumin permeation of vessels in most other tissues did not differ for controls and diabetics. These observations indicate that the functional integrity of new vessels formed in the diabetic milieu is impaired: (1) to a greater extent than that of older vessels formed before induction of diabetes and (2) relative to new vessels in nondiabetics. The implication of these observations is that molecular constituents of vessels synthesized in the diabetic milieu are quantitatively and/or qualitatively abnormal and/or their incorporation into vessels is defective.

Increased collagen cross-linkages in experimental diabetes: reversal by beta-aminopropionitrile and D-penicillamine.

The effects of diabetes on collagen cross-link formation and solubility were investigated in granulation tissue collagen induced by polyester fabric implanted subcutaneously in rats at the same time diabetes was produced by injection of streptozotocin. Thus, all the collagen analyzed was formed in a diabetic milieu. Ten days later the implants were removed and the total collagen content as well as the fraction soluble in 0.5 M acetic acid was determined. Predominantly type I collagen accumulated in the implants. Total collagen content was the same in diabetics and controls; however, the acid-soluble fraction in diabetic animals was only half that of controls (8.5% and 17.7%, respectively), and the ratio of beta chains to alpha chains in the acid-soluble fraction was higher in diabetics (0.89) than in controls (0.69). In animals treated with beta-aminopropionitrile or D-penicillamine the acid-soluble fraction of collagen from diabetics equaled that from controls. These observations indicate that both intramolecular and intermolecular cross-links are increased in type I collagen from diabetic animals. Since these cross-links interfere with degradation of collagen by collagenase, they may contribute to accelerated intimal sclerosis of arteries and to capillary basement membrane thickening in diabetes.

Thin muscle capillary basement membranes in myotonic dystrophy.

Muscle capillary basement membrane width (MCBMW) was measured in 18 myotonic dystrophy patients and compared with that in age- and sex-matched normal and diabetic subjects. The MCBMW in myotonic dystrophy patients (773 +/- 258 A) was significantly thinner than in normal subjects (925 +/- 181 A, P less than 0.05) or in diabetics (1224 +/- 614 A, P less than 0.01). An increase in MCBMW with advancing age was present in all groups but was greatest in the myotonic dystrophy groups (r = +0.59, P less than 0.01). There was no relation between MCBMW and either the degree of glucose intolerance or insulin hypersecretion in the myotonic dystrophy group, though none had fasting hyperglycemia. This is the first report of a condition associated with thinner-than-normal capillary basement membrane.

Quantitative vitreous fluorophotometry. A sensitive technique for measuring early breakdown of the blood-retinal barrier in young diabetic patients.

Vitreous and aqueous humor fluorescein concentrations were measured one hour after graded intravenous fluorescein was given to 20 juvenile diabetics, ages 20 to 40, with and without retinopathy, and to 12 controls of similar age. Vitreous fluorescein concentrations were significantly higher in diabetics, indicating breakdown of the blood-retinal barrier. Mean vitreous fluorescein values were 10.66 +/- 0.65 for the diabetics and 4.28 +/- 0.37 ng./ml. for the controls. Breakdown of the blood-retinal barrier was also confirmed in diabetics under the age of 20 without retinopathy. The blood-aqueous barrier was similarly altered in diabetics. Vitreous fluorophotometry quantitatively measures breakdown of the blood-retinal barrier, possibly the earliest detectable ocular vascular abnormality in juvenile diabetic patients.

Hemoglobin AIc as an indicator of the degree of glucose intolerance in diabetes.

Hemoglobin AIc concentration, fasting blood sugar, response to an oral glucose tolerance test, and skeletal muscle capillary basement membrane thickness were measured in diabetic patients. Hemoglobin AIc concentration correlates with both response to a glucose tolerance test (r = 0.82, p less than 0.001) and fasting blood sugar (r = 0.62, p less than 0.001). The correlation of hemoglobin AIc concentration with glucose tolerance is independent of fasting blood sugar concentration (partial r = 0.61, p less than 0.005), whereas that of hemoglobin AIc with fasting blood sugar probably reflects the relationship between fasting blood sugar levels and glucose tolerance (partial r = 0.22, p less than 0.05). Hemoglobin AIc levels do not correlate with basement membrane thickness ( r = 0.15, p less than 0.05).

Monozygotic triplets with discordance for diabetes mellitus and diabetic microangiopathy.

A set of monozygotic triplets (PE.K., P.K., S.K.) has been studied. There is no diabetes in first-degree relatives. PE.K. developed insulin-requiring (60 U. NPH) diabetes at the age of 13 years. Over a period of 11 years since that time, numerous studies of insulin and growth-hormone secretion were performed on P.K. and S.K., including multiple oral glucose tolerance tests (OGTTs), cortisone-primed oral glucose tolerance tests (C-OGTTs), intravenous glucose tolerance tests (IVGTTs), and intravenous tolbutamide tests (IVTTs). The results of each test were compared with age- and sex-matched control subjects. P. K. developed insulin-requiring (56 U. NPH) diabetes after remaining discordant for eight years. Glucose, insilin, and growth-hormone responses during all tests were normal except during the IVGTT performed four months prior to the onset of diabetes. This last IVGTT revealed a glucose disappearance rate of 0.98 per cent per minute, and the slope of the regression line of serum-insulin response (IRI) on blood glucose (BG) was markedly decreased to 0.005 micronU./ml. IRI/mg./dl. BG (controls 0.340 +/- 0.04; mean +/- S.E.M.). The insulin responses in P.K. and S.K. were similar during all OGTTs, C-OGTTs, and IVTTs. S.K. has continued to maintain normal glucose tolerance and normal insulin and growth-hormone responses during all tests. The histocompability antigen studies have revealed HLA-A2, AW24, BW15, and BW40 phenotype in these monozygotic triplets. Muscle capillary basement membranes of the nondiabetic triplet were normal, whereas both diabetic triplets manifested evidence of capillary basement membrane thickening. The clinical and biochemical profiles in these triplets and the capillary basement membrane data lend strong credence to the role of "nongenetic" determinants in the development of "genetic" diabetes as well as diabetic microangiopathy in juvenile-onset-type diabetes.

Effect of rapid normalization of plasma glucose levels on microvascular dysfunction and polyol metabolism in diabetic rats.

Effects of rapid normalization of plasma glucose levels (by insulin infused via Alzet pumps implanted intraperitoneally) on plasma insulin-like growth factor I (IGF-I) levels, granulation tissue polyol levels, and vascular permeation by 125I-labeled albumin were examined in male Sprague-Dawley rats with streptozocin-induced (60-65 mg/kg) diabetes. Two days after implantation of pumps, plasma insulin levels were twice normal levels and remained elevated (1.4-2.5 times normal) throughout the remainder of the study. Plasma glucose levels and granulation tissue polyol levels were normalized within 2 days after initiation of insulin treatment. Plasma IGF-I levels were significantly increased (2 times) by 2 days, but were not normalized until 7 days. In contrast, 125I-albumin permeation normalized at a much slower relatively linear rate and was still not completely normal after 14 days of insulin treatment. In view of 1) previous studies demonstrating that diabetes-induced increases in 125I-albumin permeation in this tissue are linked to increased metabolism of glucose to sorbitol and 2) the rapid normalization of tissue polyol levels in this study, the relatively linear rate of normalization of vascular permeability over 14 days in these studies suggests that impaired vascular barrier functional integrity in this model is mediated by structural and/or functional vascular alterations associated with sustained increased polyol metabolism rather than by increased polyol levels per se and/or by readily reversible functional and metabolic alterations associated with acute increases in polyol metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors.

This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on 1) regional blood flow (assessed with 15-microns 85Sr-labeled microspheres) and vascular permeation by 125I-labeled bovine serum albumin (BSA) and 2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic changes. We hypothesize that 1) increases in blood flow may reflect impaired contractile function of smooth muscle cells in resistance arterioles and 2) increases in vascular 125I-BSA permeation and urinary albumin excretion reflect impaired vascular barrier functional integrity in addition to increased hydraulic conductance secondary to microvascular hypertension associated with decreased vascular resistance.

Myocyte contracture, vascular resistance, and vascular permeability after global ischemia in isolated hearts from alloxan-induced diabetic rabbits.

Coronary vascular hemodynamics, albumin permeation, and myocyte contractility were assessed in isolated hearts from 6-mo alloxan-induced diabetic (ALX-D) rabbits during 3 h of reperfusion after 40 min of global no-flow ischemia. Residue-detection data, generated during the single passage of a bolus of 125I-labeled bovine serum albumin (125I-BSA) through the coronary vasculature, were used to estimate indices of vascular function, including the mean transit time of 125I-BSA, the fractional rate of intravascular clearance of 125I-BSA, and 125I-BSA permeation of coronary vessels. During reflow after ischemia in hearts from control rabbits, vascular resistance increased approximately three times that at baseline, left ventricular end-diastolic pressure (LVEDP) increased 8-10 times, and maximum +dP/dt recovered 0.4 times baseline, whereas the fractional rate of washout of intravascular 125I-BSA decreased to less than one-half of baseline values (was prolonged 2-fold), and albumin permeation and mean-transit time were increased 3 and 5 times baseline, respectively. In hearts from diabetic rabbits, vascular resistance was similar to the control group before ischemia but increased only one-third as much during reflow after ischemia. Increases in LVEDP during reflow were approximately 50% lower than controls, and +dP/dt recovered approximately 2.5 times more than in control hearts. 125I-BSA permeation in diabetics was similar to controls before ischemia, but during reflow increased 6 times (approximately 2 times controls). Washout of intravascular 125I-BSA was prolonged approximately 20% versus baseline during 3 h of reflow in hearts from diabetic rabbits. Thus, ALX-D in the rabbit delayed ischemia-reperfusion injury to myocytes and vascular smooth muscle cells while increasing vascular albumin permeation.

Sex steroid dependency of diabetes-induced changes in polyol metabolism, vascular permeability, and collagen cross-linking.

The effects of castration on diabetes-induced increases in collagen cross-linking and vascular permeability and on polyol levels in new granulation tissue formed after induction of streptozocin (STZ) diabetes were examined in male Sprague-Dawley rats. New granulation tissue formation was induced by implanting sterile polyester fabric subcutaneously (s.c.) at the time of STZ injection 3 wk before assessment of vascular permeability and collagen cross-linking. Castration was performed 10 days before implanting the fabric. The characteristic increases in collagen cross-linking (manifested by decreased solubility in 0.5 M acetic acid) and in albumin permeation of the vasculature seen in intact diabetic rats were completely prevented by castration. Net collagen accumulation was not affected by diabetes or castration. Castration also markedly diminished diabetes-induced increases in tissue levels of sorbitol and completely prevented the decreases in tissue levels of myo-inositol and scyllo-inositol observed in intact diabetic rats, but had no effect on serum glucose levels, nonenzymatic glycosylation of plasma and granulation tissue proteins, or plasma somatomedin-C levels. The demonstration that castration prevents diabetes-induced increases in vascular permeability and collagen cross-linking as well as imbalances in tissue levels of sorbitol, myo-inositol, and scyllo-inositol in this model indicates that all of these changes are sex steroid-dependent phenomena. While the pathogenesis of these vascular permeability and collagen cross-linking changes is clearly multifactorial, these new findings: indicate that the role of sex steroids in the development of late complications of diabetes may be far more important than hitherto suspected, and suggest an explanation for the clinical observation that diabetic complications are uncommon in prepubertal diabetic subjects regardless of duration of diabetes.

Modulation of hemodynamic and vascular filtration changes in diabetic rats by dietary myo-inositol.

To assess the potential of myo-inositol-supplemented diets to prevent diabetes-induced vascular functional changes, we examined the effects of diets supplemented with 0.5, 1, or 2% myo-inositol on blood flow and vascular filtration function in nondiabetic control rats and rats with streptozocin-induced diabetes (STZ-D). After 1 mo of diabetes and dietary myo-inositol supplementation, 1) 131I-labeled bovine serum albumin (BSA) permeation of vessels was assessed in multiple tissues, 2) glomerular filtration rate (GFR) was estimated as renal plasma clearance of 57Co-labeled EDTA, 3) regional blood flows were measured with 15-microns 85Sr-labeled microspheres, and 4) endogenous albumin and IgG urinary excretion rates were quantified by radial immunodiffusion assay. In STZ-D rats, 131I-BSA tissue clearance increased significantly (2- to 4-fold) in the anterior uvea, choroid-sclera, retina, sciatic nerve, aorta, new granulation tissue, diaphragm, and kidney but was unchanged in skin, forelimb muscle, and heart. myo-Inositol-supplemented diets reduced diabetes-induced increases in 131I-BSA clearance (in a dose-dependent manner) in all tissues; however, only in new granulation tissue and diaphragm did the 2% myo-inositol diet completely normalize vascular albumin permeation. Diabetes-induced increases in GFR and in urinary albumin and IgG excretion were also substantially reduced or normalized by dietary myo-inositol supplements. Increased blood flow in anterior uvea, choroid-sclera, kidney, new granulation tissue, and skeletal muscle in STZ-D rats also was substantially reduced or normalized by the 2% myo-inositol diet. myo-Inositol had minimal if any effects on the above parameters in control rats. These observations indicate that diabetes-induced increases in regional blood flow, 131I-BSA permeation, GFR, and urinary protein excretion can be markedly reduced or normalized by consumption of myo-inositol-supplemented diets that raise plasma myo-inositol levels approximately fivefold. The failure of the 2% myo-inositol diet to normalize GFR and blood flow and albumin permeation in several tissues despite markedly elevated plasma myo-inositol levels and normal or elevated tissue myo-inositol levels indicates that if vascular functional changes in these tissues are linked to altered myo-inositol levels, they are resistant to normalization by elevation of plasma myo-inositol levels. These results suggest that other factors independent of changes in relative or absolute tissue myo-inositol levels may play an important role in the pathogenesis of diabetes-induced vascular functional changes in these tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased vascular permeability in spontaneously diabetic BB/W rats and in rats with mild versus severe streptozocin-induced diabetes. Prevention by aldose reductase inhibitors and castration.

125I-labeled albumin permeation (IAP) has been assessed in various tissues in spontaneously diabetic insulin-dependent female BB/W rats and in male Sprague-Dawley rats with severe or mild forms of streptozocin-induced diabetes (SS-D and MS-D, respectively). In BB/W diabetic rats and in rats with SS-D, indices of IAP were significantly increased in tissues and vessels predisposed to diabetic vascular disease in humans, including the eyes (anterior uvea, posterior uvea, and retina), sciatic nerve, aorta, kidney, and new vessels formed after induction of diabetes. No evidence of increased IAP was observed in heart, brain, testes, or skeletal muscle in BB/W or SS-D rats. In MS-D rats, indices of IAP were increased only in the kidney and in new vessels formed after the onset of diabetes. Marked tissue differences were observed in the effects of two structurally different aldose reductase inhibitors (sorbinil and tolrestat) and of castration on diabetes-induced increases in IAP and in tissue levels of polyols in SS-D rats. Both aldose reductase inhibitors and castration completely prevented diabetes-induced increases in IAP in new vessels and in sciatic nerve in BB/W and SS-D rats. Both aldose reductase inhibitors also markedly decreased IAP in the anterior uvea (approximately 85%), posterior uvea (approximately 65-75%), retina (approximately 65-70%), and kidney (approximately 70-100%); castration reduced IAP in the anterior uvea (approximately 55%), kidney (approximately 50%), and retina (approximately 30%) but had no effect on the posterior uvea. The diabetes-induced increases in IAP in the aorta were reduced only slightly (approximately 20%) by aldose reductase inhibitors and castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Sorbinil prevents diabetes-induced increases in vascular permeability but does not alter collagen cross-linking.

In recent studies we have demonstrated a marked increase in albumin permeation of new vessels formed by angiogenesis (in subcutaneous tissue) in the diabetic milieu. Likewise, lysyl oxidase-mediated collagen cross-linking is markedly increased in the scar tissue associated with angiogenesis. The present studies were undertaken to determine whether sorbinil, a chemical inhibitor of aldose reductase that has been shown to prevent and reverse diabetic cataracts and neuropathy, also could prevent the vascular permeability and collagen cross-linking changes in this model. Vascular permeation by 125I-BSA, collagen cross-linking, and tissue levels of sorbitol, myo-inositol, and scyllo-inositol were assessed in male Sprague-Dawley rats 3 wk after injection of streptozocin and induction of angiogenesis and collagen synthesis in polyester fabric implanted subcutaneously. Sorbinil (approximately 25 mg/kg/day) added to the diet of diabetic rats reduced the diabetes-induced increases in albumin permeation by 80%, completely prevented diabetes-induced changes in tissue levels of sorbitol and myo-inositol, and markedly reduced diabetes-induced changes in tissue levels of scyllo-inositol. In contrast, sorbinil had no effect on plasma glucose levels or collagen solubility (an index of collagen cross-linking). These observations indicate that increased vascular permeability associated with diabetes is linked to imbalances in sorbitol/inositol metabolism. These findings also indicate that diabetes-induced increases in vascular permeability and in collagen cross-linking are independent phenomena and diabetes-induced increases in vascular permeability are largely preventable by treatment with an aldose reductase inhibitor in the face of high plasma glucose levels.

Hyperglycemic pseudohypoxia and diabetic complications.

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADH/NAD+, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation.