Serum caeruloplasmin as a coronary risk factor in the elderly: the Rotterdam Study.

Serum Cu and caeruloplasmin levels have been suggested to be independent risk factors for CHD operating through oxidative modification of LDL. However, given its function as an acute-phase protein, the question has been raised whether an elevated caeruloplasmin level is not merely an indicator of inflammation. In the current study, we investigated whether serum caeruloplasmin was associated with subsequent myocardial infarction, taking into account indices of inflammation. The study population consisted of 210 cases of first myocardial infarction and controls, frequency-matched on age (5-year categories) and sex, selected from the population-based cohort of the Rotterdam Study. Serum caeruloplasmin levels were significantly elevated in cases of myocardial infarction compared with controls (510 (SD 110) v. 470 (SD 100) mg/l; P = 0.007). Risk of myocardial infarction for the highest compared with the lowest quartile of caeruloplasmin was 2.46 (95% CI 1.04, 6.00; Ptrend = 0.043) after adjustment for age, sex, BMI, pack-years smoked, serum cholesterol, systolic blood pressure, and income. The relative risk was most evident in current smokers. Adjustment for C-reactive protein and leucocyte count reduced the excess risk by 33%. This suggests that a substantial part of the observed association between serum caeruloplasmin and CHD may be attributed to inflammation processes rather than to the pro-oxidant activity of caeruloplasmin.

Serum ferritin and risk of myocardial infarction in the elderly: the Rotterdam Study.

BACKGROUND: Elevated body iron stores have been suggested to be a risk factor for ischemic heart disease. OBJECTIVE: We examined whether elevated serum ferritin concentrations, other indicators of iron status, and dietary iron affected the incidence of myocardial infarction (MI) in an elderly population. DESIGN: A nested, case-control study of 60 patients who had their first MI and 112 age- and sex-matched control subjects embedded in the population-based cohort of the Rotterdam Study. RESULTS: The age- and sex-adjusted risk of MI for subjects with serum ferritin concentrations > or = 200 microg/L was 1.82 (95% CI: 0.90, 3.69; P = 0.096). The odds ratio (OR) was 1.26 (95% CI: 0.98, 1.64; P = 0.078) for the highest tertile of serum ferritin and was only slightly altered in a multivariate model. Risk of MI associated with the highest tertile of ferritin was most evident in current or former smokers (OR: 1.68; 95% CI: 1.17, 2.47; P for trend = 0.008) and in subjects with hypercholesterolemia (OR: 1.43; 95% CI: 0.99, 2.11; P for trend = 0.056) or diabetes (OR: 2.41; 95% CI: 1.12, 7.67; P for trend = 0.027). No association with risk of MI was observed for tertiles of serum iron, serum transferrin, or total dietary iron. For dietary heme iron, risk of MI was significantly increased in a multivariate model in which dietary energy, fat, saturated fat, and cholesterol were adjusted for (OR: 4.01; 95% CI: 1.17, 15.87; P for trend = 0.031). CONCLUSION: In the presence of other risk factors, serum ferritin may adversely affect ischemic heart disease risk in the elderly.

Evidence against the involvement of multiple radical generating sites in the expression of the vascular cell adhesion molecule-1.

The present study was undertaken to investigate the hypothesis that multiple oxygen radical generating systems contribute to the tumor necrosis factor (TNF) alpha-stimulated transcriptional activation of the vascular cell adhesion molecule (VCAM)-1 in endothelial cells. Experimental evidence has implicated the cytochrome P450 monooxygenase and a phagocyte type NADPH-oxidase as a source of oxygen radicals in these cells. We show here that endothelial cells exhibit cytochrome P450 activity by measuring the O-dealkylation of the exogenous substrate 7-ethoxyresorufin, but components of the phagocyte-type NADPH oxidase could not be demonstrated in endothelial cells. In that latter respect it was surprising that the NADPH oxidase inhibitor apocynin completely prevented the accumulation of VCAM-1 mRNA. However, we found that apocynin also acts as an inhibitor of cytochrome P450 activity in endothelial cells. Therefore the inhibitory effect of apocynin on the induction of VCAM-1 may no longer be used to demonstrate a role for the NADPH oxidase in this process. Furthermore, different cytochrome P450 inhibitors Co2+, metyrapone, SKF525a decreased the endothelial VCAM-1 expression stimulated by TNFalpha. Also under hypoxic conditions the expression of VCAM-1 was reduced. On this basis we assume that the oxygen dependent step in the intracellular signalling cascade underlying the TNFalpha stimulated transcriptional activation of VCAM-1 resides in the activity of a cytochrome P450 dependent monooxygenase. The finding that the phospholipase A2 inhibitor bromophenacylbromide inhibited the expression of VCAM-1 may indicate that arachidonic acid serves as a substrate for the cytochrome P450 monooxygenase reaction, but further research is needed to elucidate the particular cytochrome P450 family member mediating the expression of VCAM-1.

Role of interleukin 1 and granulocyte colony-stimulating factor in photofrin-based photodynamic therapy of rat rhabdomyosarcoma tumors.

Neutrophils play an important role in the efficacy of photodynamic therapy (PDT). These leukocytes rapidly accumulate into the tumor lesion after PDT and most likely eradicate the remaining attenuated tumor cells. The underlying mechanism of the accumulation of neutrophils at the time of PDT is not known. Therefore, we determined the effect of PDT on the course of mature and immature neutrophils in the circulation of rhabdomyosarcoma-bearing rats and studied the changes in the level of interleukin (IL)-1beta as an important stimulator of the proliferation of precursor cells of the granulocyte lineage in the bone marrow. We found that the effect of PDT on tumor growth was preceded by a rapid and specific increase of the number of mature neutrophils in the peripheral blood as early as 4 h after the start of PDT treatment and reaching maximum values after 8 h. At 24 h, the neutrophil numbers in the PDT-treated rats were still elevated as compared to sham-treated rats. In sham-treated rats, the numbers of blood monocytes and lymphocytes decreased by about 50% after 2 h and returned to their normal levels as soon as 2 h later. In PDT-treated rats, the course of monocyte numbers showed a similar pattern; however, lymphocyte numbers did not reach the normal range until 24 h. The specific increment of neutrophils was preceded by an increase of band neutrophil numbers and elevated serum levels of IL-1beta which were maximal at 2 h after the start of PDT. Pearson correlation analysis showed a significant association between the serum levels of IL-1beta at this time point and the number of band neutrophils at 4 h (R2 = 0.58; P = 0.03) and the number of mature neutrophils at 8 h (R2 = 0.54; P = 0.04). This suggests that PDT evoked an IL-1-dependent increased production rate of neutrophils in the bone marrow. Further investigation showed that the injection of anti-granulocyte colony-stimulating factor (G-CSF) antibodies not only attenuated the increase in neutrophil numbers but also greatly decreased the efficacy of PDT. On this basis, we suppose that an IL-1-induced release of G-CSF by PDT underlies this nonspecific immune reaction to the tumor. Apparently, G-CSF not only stimulates the production rate of neutrophils in the bone marrow but also increases the functional activity of these leukocytes to become indispensable tumor cell killers.

p38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level.

The cytokine tumor necrosis factor (TNF) alpha was found to stimulate the p38 mitogen activated protein (MAP) kinase signalling cascade in human umbilical vein endothelial cells. TNFalpha increased the activity of the p38 substrate MAP kinase-activated-protein (MAPKAP) kinase 2 and the subsequent phosphorylation of the small heat shock protein Hsp27 about two to three fold. This stimulation was blocked almost completely by the specific p38 MAP kinase inhibitor SB203580. This inhibitor also suppressed the TNFalpha-induced surface expression of the endothelial adhesion molecule vascular cell adhesion molecule (VCAM)-1. In contrast, inhibition of p38 MAP kinase had no effect on the stimulated surface expression of the intercellular cell adhesion molecule (ICAM)-1. VCAM-1 mRNA accumulation induced by TNFalpha was not affected by SB203580, suggesting that the p38 MAP kinase signalling cascade regulates the endothelial expression of VCAM-1 at the post-transcriptional level.

Evidence for an important role of neutrophils in the efficacy of photodynamic therapy in vivo.

To investigate the role of neutrophils in the efficacy of photodynamic therapy (PDT) in rhabdomyosarcoma-bearing rats, the number of these circulating phagocytes was decreased or increased before interstitial PDT by use of rabbit anti-rat neutrophil serum or granulocyte colony-stimulating factor, respectively. After administration of the antiserum, the number of circulating neutrophils decreased by 99.9%. However, the number of monocytes, lymphocytes, and platelets decreased as well (by 100%, 80%, and 25%, respectively). Under these conditions, PDT did not retard tumor growth at all. However, after cessation of the antiserum treatment 5 days after PDT, a striking decrease in the growth rate occurred subsequent to an increase above the normal range of the number of circulating neutrophils. Administration of the granulocyte colony-stimulating factor led to a specific 4-fold increase in the number of circulating neutrophils. In these rats, the tumor growth at day 2 after PDT was retarded as compared with PDT-treated rats that received saline only. Statistical evaluation of both experimental conditions showed that the efficacy of PDT, expressed as the percentage of change in tumor volume at day 2 after treatment, was dependent on the number of circulating neutrophils present at the day of PDT (P = 0.001; r2 = 0.482). Apparently, neutrophils are indispensable for successful PDT in vivo.

Photodynamic treatment of human endothelial cells promotes the adherence of neutrophils in vitro.

The effects of photodynamic treatment (PDT) on venules include vascular leakage accompanied by oedema formation, vasoconstriction and blood flow stasis. The goal of this study was to gain insight into the mechanism underlying these vascular events by studying one of the earliest observations after PDT, granulocyte adhesion, in an in vitro model. For this purpose human umbilical vein endothelial cells (HUVECs) preincubated with Photofrin II (PII) were illuminated with red light and incubated with neutrophils. PDT led to a dramatic change in the morphology of the endothelial cells. Clearly, neutrophils adhered to the subendothelial matrix and their adherence coincided with an increase in the percentage of exposed subendothelial matrix by the gradual contraction of endothelial cells. Furthermore, the increase in adherence was dependent on drug dose, illumination time and the time delay after PDT. The neutrophil adherence could be inhibited by anti-beta2-integrin antibodies, which suggests that the alphaL-, alphaM- or alphaX-beta2 receptors of the neutrophil mediated this phenomenon. At 4 degrees C or by preincubation of the neutrophils with staurosporin, their adherence to the subendothelial matrix exposed by PDT of endothelial cells could be prevented. Apparently, activation of the beta2-integrin receptor by interaction with the subendothelial matrix is necessary for the increased binding of neutrophils. Taken together, these in vitro findings suggest that the PDT-induced contraction of the endothelial cells permits neutrophil adherence to the subendothelial matrix. It is conceivable that a similar mechanism contributes to the initial adherence of granulocytes to the vessel wall as observed after PDT in vivo.

Prevention of late lumen loss after coronary angioplasty by photodynamic therapy: role of activated neutrophils.

Restenosis after coronary angioplasty arises from fibrocellular intimal hyperplasia and possibly failure of the artery to enlarge adequately. Which mechanisms underlie this process is only partly understood. No drugs have been clinically effective in reducing the incidence of restenosis. Since recently, photodynamic therapy (PDT) is being investigated as a possible treatment for intimal hyperplasia. PDT involves the systemic administration of a light-excitable photosensitizer that is taken up rather preferentially by rapidly proliferating cells. During laser irradiation light energy is transferred from the photosensitizer to oxygen generating the highly reactive singlet oxygen. This potent oxidizer can cause severe cellular damage. After PDT of a balloon-injured artery from the rat and rabbit the media remained acellular for several weeks to months, and intimal hyperplasia did not occur. The endothelial lining regenerated by two weeks, but why smooth muscle cells did not repopulated the media is not known. Neutrophils seem to play an important role in the prevention of restenosis after coronary angioplasty, since the activation status of this type of phagocyte is directly related to vessel diameter at late follow-up. Furthermore, it has been observed that neutrophils adhere to the microvascular wall upon PDT in vivo. In vitro findings suggest that the increased neutrophil adherence was not dependent on a decreased release of the anti-adhesive factors NO and prostacyclin by the PDT-treated endothelial cells. Furthermore, PDT did not stimulate the expression of P-selectin by the endothelial cells, one of the adhesion receptors for neutrophils. The endothelial cells only retract upon PDT allowing the adherence of neutrophils by their beta 2-integrin adhesion receptors to the subendothelial matrix. On the basis of these findings, we presume that the successful prevention of intimal hyperplasia by PDT partly depends on the presence of the neutrophil at the site of the lesion.

Manganese-induced hydroxyl radical formation in rat striatum is not attenuated by dopamine depletion or iron chelation in vivo.

The present studies were aimed at investigating the possible roles of dopamine (DA) and iron in production of hydroxyl radicals (OH) in rat striatum after Mn2+ intoxication. For this purpose, DA depletions were assessed concomitant with in vivo 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formation from the reaction of salicylate with OH, of which 2,3-DHBA is a nonenzymatic adduct. Following intrastriatal Mn2+ injection, marked 2,3-DHBA increases were observed in a time- and dose-dependent fashion reaching maximum levels at 6-18 h and a plateau beyond 0.4 micromol (fourfold increase). The delayed increase of 2,3-DHBA levels suggestS that Mn2+ induces OH formation in the living brain by an indirect process. The early DA depletion (2 h) and relatively late OH formation (6 h) indicate independent processes by Mn2+. In addition, depletion of DA (about 90%) by reserpine pretreatment not significantly alter Mn2+-induced 2,3-DHBA formation or the extent of DA depletion, suggesting that DA or DA autoxidation are not participating in Mn2+-induced OH formation in vivo. Furthermore, Mn2+ injection did not significantly alter the low molecular weight weight iron pool in striatum, and co-injections of the iron-chelator deferoxamine with Mn(2+) into striatum did not significantly attenuate Mn(2+)-induced 2,3-DHBA formation. These findings suggest no role of chelatable iron in generation of Mn(2+)-induced OH, but do not exclude a role for mitochondrial heme-iron or peroxynitrite (Fe-indepeNdent) in Mn2+-induced OH formation.

Late lumen loss after coronary angioplasty is associated with the activation status of circulating phagocytes before treatment.

BACKGROUND: The purpose of this pilot study was to identify biological risk factors for restenosis after percutaneous transluminal coronary angioplasty (PTCA) to predict the long-term outcome of PTCA before treatment. METHODS AND RESULTS: To investigate whether blood granulocytes and monocytes could determine luminal renarrowing after PTCA, several characteristics of these phagocytes were assessed before angioplasty in 32 patients who underwent PTCA of one coronary artery and who had repeat angiograms at 6-month follow-up. The plasma levels of interleukin (IL)-1 beta, tumor necrosis factor-alpha, IL-6, fibrinogen, C-reactive protein, and lipoprotein(a) before angioplasty were assessed as well. We found that the expression of the membrane antigens CD64, CD66, and CD67 by granulocytes was inversely associated with the luminal renarrowing normalized for vessel size (relative loss) at 6 months after PTCA, while the production of IL-1 beta by stimulated monocytes was positively associated with the relative loss. Next, these univariate predictors were corrected for the established clinical risk factors of dilation of the left anterior descending coronary artery and current smoking, which were statistically significant classic predictors in our patient group. Only the expression of CD67 did not predict late lumen loss independent of these established clinical risk factors. Multiple linear regression analysis showed that luminal renarrowing could be predicted reliably (R2 = .65; P < .0001) in this patient group on the basis of the vessel dilated and only two biological risk factors that reflect the activation status of blood phagocytes, ie, the expression of CD66 by granulocytes and the production of IL-1 beta by stimulated monocytes. CONCLUSIONS: The results of the present study indicate that activated blood granulocytes prevent luminal renarrowing after PTCA, while activated blood monocytes promote late lumen loss. To validate this new finding, further study in an independent patient group is required.

Effect of hypoxia on adherence of granulocytes to endothelial cells in vitro.

The objective of this study was to investigate the effect of hypoxia on the adhesiveness of endothelial cells for granulocytes. Human umbilical vein endothelial cells (HUVEC) were exposed to a PO2 of 7.5 mmHg (1.0 kPa), and the adherence of granulocytes was assessed under continuous hypoxia by means of a hypoxic incubator room. After 2 h of hypoxia the adherence of granulocytes decreased to 50% of the normoxic control, which was not due to a decreased viability of the endothelial cells nor to an increased generation of the antiadhesive factors nitric oxide, prostacyclin, and adenosine. Hypoxia also had no effect on the expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 on the endothelium. Although the mechanism of the action of hypoxia on the adhesiveness of endothelial cells remains unclear as yet, our data suggest that HUVEC possess a protective mechanism that prevents granulocyte adherence to endothelial cells under extreme hypoxic conditions. The decreased adherence seems paradoxical to the in vivo situation for which the increased margination of granulocytes within the vascular compartment of the ischemic tissue has been observed. However, hypoxia did not impair the potential adhesiveness of HUVEC, since stimulation of endothelial cells under hypoxic conditions with calcium ionophore or lipopolysaccharide increased the adherence of granulocytes in a similar fashion as under normoxic conditions. We therefore conclude that the increased margination of granulocytes during ischemia may be accomplished by the additional stimulation of hypoxic endothelial cells.

The increased susceptibility to hydrogen peroxide of the (post-)ischemic rat heart is associated with the magnitude of the low molecular weight iron pool.

Recently we have shown that intracellular low molecular weight (LMW) iron increases during ischemia. It is hypothesized that this increase in LMW iron during ischemia underlies the reported hydrogen peroxide toxicity toward ischemic hearts. To investigate this hypothesis, rat hearts were subjected to 15 min of no-flow ischemia and reperfused with buffer saturated against 95% N2 and 5% CO2 (anoxic reperfusion) for 7 min. Hearts were then switched to buffer saturated against 95% O2 and 5% CO2 (reoxygenation) to assess functional recovery. The cardiac function recovered to 80 +/- 7% of the preischemic value. When the anoxic reperfusion was applied in the presence of 10 microM hydrogen peroxide, functional recovery after reoxygenation was 47 +/- 7%. Hearts that were perfused with deferoxamine before ischemia and then subjected to ischemia and anoxic reperfusion in the presence of 10 microM hydrogen peroxide recovered to 78 +/- 8%. Immediate reoxygenation after ischemia led to only 45 +/- 6% recovery of function. During ischemia, LMW iron increased from 49 +/- 45 to 183 +/- 45 pmol/mg protein (p < .05) and decreased to 58 +/- 38 pmol/mg protein (p < .05) during the subsequent anoxic perfusion. Rat hearts preloaded with deferoxamine showed a slightly higher LMW iron content than normal (85 +/- 23 and 49 +/- 45 pmol/mg protein, respectively; n.s.), which showed a small, nonsignificant increase up to 136 +/- 42 pmol/mg protein after 15 min of ischemia. No significant changes were found in reduced and oxidized glutathione content and glutathione peroxidase or catalase activities under those conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Malondialdehyde and glutathione production in isolated perfused human and rat hearts.

A number of studies show the relation between oxygen-derived free radicals and cardiac ischemia/reperfusion injury. However, little is known about oxidative stress in the human heart, which can be measured by oxidized glutathione (GSSG) and malondialdehyde (MDA) formation. Furthermore, data on MDA production by rat hearts are controversial, possibly because of the use of the aspecific thiobarbituric acid assay. Therefore, GSSG and MDA were measured, with colorimetric and high-performance liquid chromatographic assays, respectively, in buffer-perfused explanted human hearts and normal rat hearts made temporarily ischemic. Human hearts received cardioplegia; rat hearts were studied in a control and an ischemic group with or without cardioplegia. Baseline GSSG release was < 0.01 nmol.min-1.g wet wt-1 in both species. During reperfusion, GSSG release from human hearts and from ischemic and cardioplegic/ischemic rat hearts peaked at 0.24 +/- 0.12, 1.1 +/- 0.4, and 0.19 +/- 0.04 nmol.min-1.g-1, respectively. MDA was undetectable (< 0.02 nmol.min-1.g-1) in the effluent of both species and in human hearts (< 4 nmol/g protein). Rat heart reduced glutathione levels decreased 32% as a consequence of cardioplegia and ischemia. Cardioplegia induced a 41% (P = .08) decrease in rat heart MDA content, whereas cumene hydroperoxide increased it 3.6 times (P < .01). Thus, after ischemia human and rat hearts release GSSG, indicating that oxidative stress has occurred. Apparently, lipid peroxidation takes place in normal rat hearts, decreases after cardioplegia, but does not increase after ischemia/reperfusion. Human hearts lack MDA under normoxic and ischemic conditions. This novel finding seems to reflect a low MDA-forming potential in both situations.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of desferrioxamine on iron metabolism and lipid peroxidation in hepatocytes of C57BL/10 mice in experimental uroporphyria.

The effects of the iron chelator desferrioxamine (DFx) on liver iron accumulation, malondialdehyde (MDA) production, porphyrin accumulation and uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37) activity were investigated over a period of 14 weeks in C57BL/10 mice, made porphyric by the administration of hexachlorobenzene (HCB) and iron-dextran (Imferon, IMF) or IMF alone. In addition, we measured the amount of low molecular weight (LMW) iron in liver tissue to determine a possible correlation with MDA production. These experiments showed that combined treatment with HCB + IMF, as well as IMF alone, resulted in porphyrin accumulation, increased MDA production and reduced URO-D activity, whereas HCB alone had no effect. DFx caused a reduction in hepatic porphyrins, this reduction being more distinct in the IMF group than in the HCB + IMF group. The effect of DFx on MDA production and URO-D activity was in agreement with the results on porphyrin accumulation. LMW iron pool measurements at 11 weeks correlated well with data on MDA production in all treated groups in that period (r2 = 0.84), suggesting both variables are interdependent. In conclusion, these results suggest an important role for iron in porphyrin accumulation, probably through its catalytic role in the generation of oxygen-related free radicals, resulting in direct damage to URO-D. The effectiveness of DFx in reducing porphyrin accumulation is probably the result of a reduction in LMW iron, thus diminishing the amount of iron available for a catalytic role in the generation of oxygen-related free radicals.

Effect of photodynamic therapy on the endothelium-dependent relaxation of isolated rat aortas.

The early vascular effects of photodynamic therapy (PDT) include transient vasoconstriction and platelet aggregation. Since endothelium-derived relaxing factor (EDRF) is a potent vasodilator and inhibitor of platelet aggregation, we questioned whether PDT impairs the production of EDRF. To study this possible effect of PDT, endothelium-dependent relaxations of thoracic aortas obtained from male Wistar rats were determined. The aortic rings were connected to a isometric force transducer, exposed to various doses of Photofrin porfimer sodium (Photofrin) (0.1-1.0 microgram/ml), and illuminated with red light (wavelength > 610 nm, 14.6 +/- 1.5 mW/cm2) for different time periods (5-25 min). Endothelium-dependent relaxation was induced by acetylcholine in precontracted aortic rings. This EDRF-mediated relaxation was decreased after PDT in a light dose- and drug dose-dependent manner. Light microscopic examination did not show loss of endothelial cells. Similar results were obtained with rat aortas exposed to Photofrin in vivo and illuminated in vitro. Direct smooth muscle relaxation induced with sodium nitroprusside was not impaired, showing that PDT did not reduce the ability of smooth muscles to relax. No effect on the contractile responses was found either. We conclude that PDT impairs the production or release of EDRF by the endothelium. This could play an important role in the initial events occurring in vivo during and after PDT.

In vitro and ex vivo xanthine oxidoreductase activity in rat and guinea-pig hearts using hypoxanthine or xanthine as substrate.

Through oxyradical formation xanthine oxidoreductase (XOD) could play a role in the etiology of cardiac damage. Its measurement poses problems, due to little substrate specificity, self-inactivation and endogenous inhibitors. Perfusion of guinea-pig hearts with hypoxanthine gave rise to only little xanthine release; in contrast rat hearts showed vivid xanthine production. Therefore, xanthine breakdown was hypothesized to exceed its formation in guinea-pig hearts. The kinetics of both substrates for XOD in cardiac homogenates were therefore compared with those obtained in perfused hearts. Oxypurine contents and effluent catabolites were determined by HPLC. Regardless of substrate, Vmax values in homogenates were about 38 and 13 mU/g for rat and guinea-pig heart, respectively. Km values were in the 3-5 microM range; therefore the hypothesis concerning the low xanthine release in guinea-pig hearts must be rejected. Activities in hearts perfused with hypoxanthine (50 microM) were 40 and 18 mU/g for rat and guinea pig, respectively; perfusion with xanthine produced < 50% of the activities observed with hypoxanthine (p < 0.002). Intracellular xanthine concentration, estimated from sorbitol distribution space and myocardial xanthine content was negative in both species, contrasting intracellular hypoxanthine levels, which approached extracellular concentrations. This disparate distribution indicates that hypoxanthine transport across the cell membrane far exceeds that of xanthine. Consequently, hypoxanthine is preferable to xanthine as substrate in perfused hearts to estimate XOD activity in situ.