Lipid peroxidation and antioxidants status in human malignant and non-malignant thyroid tumours.

INTRODUCTION: Thyroid epithelial cells produce moderate amounts of reactive oxygen species that are physiologically required for thyroid hormone synthesis. Nevertheless, when they are produced in excessive amounts, they may become toxic. OBJECTIVE: The present study is aimed to compare the lipid peroxidation (LPO), antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and non-protein thiols (reduced glutathione (GSH)) in human thyroid tissues with malignant and non-malignant disorders. DESIGN AND METHODS: The study used human thyroid tissues and blood samples from 157 women (147 diseased and 10 normal). Thyroid hormones, oxidative stress markers and antioxidants were estimated by standard methods. RESULTS: LPO significantly increased in most of the papillary thyroid carcinoma (PTC: 82.9%) and follicular thyroid adenoma (FTA: 72.9%) tissues, whilst in a majority of nodular goitre (69.2%) and Hashimoto's thyroiditis (HT: 73.7%) thyroid tissues, it remained unaltered. GSH increased in PTC (55.3%), remained unaltered in FTA (97.3%) and all other goiter samples studied. SOD increased in PTC (51.1%) and all other malignant thyroid tissues studied. CAT remained unaltered in PTC (95.7%), FTA (97.3%) and all other non-malignant samples (HT, MNG, TMNG) studied. GPx increased in PTC (63.8%), all other malignant thyroid tissues and remained unaltered in many of the FTA (91.9%) tissues and all other non-malignant samples (HT, MNG, TMNG) studied. CONCLUSIONS: In the case of non-malignant thyroid tumours, the oxidant-antioxidant balance was undisturbed, whilst in malignant tumours the balance was altered, and the change in r value observed in the LPO and SOD pairs between normal and PTC tissues and also in many pairs with multi-nodular goitre (MNG)/toxic MNG tissues may be used as a marker to differentiate/detect different malignant/non-malignant thyroid tumours.

Evaluation of protective effect of myricetin, a bioflavonoid in dimethyl benzanthracene-induced breast cancer in female Wistar rats.

BACKGROUND: Breast cancer is one of the most common cancers worldwide. Alarmingly, the incidence of breast cancer is rising rapidly in India. AIM: The present research was focused to assess the role of myricetin; a bioflavonoid in 7,12-dimethylbenzanthracene (DMBA)-induced breast cancer in female Wistar rats. MATERIALS AND METHODS: A total of 36 female Wistar rats (total 6 groups, n = 6 per group) 6 - 8 weeks old, weighing 150 gm were used in the study. DMBA was given at the dose of 7.5 mg/kg subcutaneously in the mammary region once a week for 4 consecutive weeks in group 2. Vincristine was given in the dose of 500 µg/kg intraperitonially every week for 4 consecutive weeks in group 3. Myricetin was given orally in a dose of 50, 100, and 200 mg/kg in group 4, 5, and 6 respectively. The statistical significance of the data was determined using one way analysis of variance and Duncan's multiple range test. RESULTS: The result showed that myricetin increased the antioxidant levels in plasma, erythrocyte lysate, and breast tissue and was effective in preventing the oxidative damage induced by the carcinogen DMBA. Myricetin 50, 100, and 200 mg/kg/oral for 120 days treated animal resulted comparable results to that of standard vincristine and control groups. CONCLUSIONS: Myricetin was found to be either equieffective or more effective than vincristine in all the parameters studied. Myricetin proved the capacity of flavonols to act as antioxidant in cells represents a potential treatment in the field of oncology.

Androgen receptor expression in human thyroid cancer tissues: a potential mechanism underlying the gender bias in the incidence of thyroid cancers.

Gender bias in the incidence of thyroid cancer is well known, however, the underlying mechanism is largely unknown. The current study determines variations in the molecular characteristics of thyroid cancers between men and women. Normal and cancerous thyroid tissues were collected from a total of 125 men and women who underwent surgical thyroidectomy. Testosterone levels in serum and thyroid cancer tissues were elevated in women while it decreased in men compared to respective control groups; whereas, ligand binding activity increased in men and decreased in women. Androgen receptor (AR) mRNA expression increased in a majority of men while it decreased in a majority of women except those with follicular thyroid carcinoma (FTC). In thyroid cancers of women, Pearson's correlation analysis showed a positive correlation of AR mRNA with AR protein, CBP and Sp1, whereas AR mRNA showed a negative correlation with p53. In case of men, AR mRNA showed a positive correlation with AR and cyclin D1 proteins in papillary thyroid carcinoma (PTC); and CBP and Sp1 in follicular thyroid adenoma (FTA), whereas AR mRNA showed a positive correlation with p53. Our study identified for the first time that AR is posttranscriptionally regulated by miR-124a in thyroid cancer tissues. Further, our in vitro studies with a PTC cell line (NPA-87-1) showed miR-124a as the potent inhibitor of AR that impairs cell proliferation even in the presence of testosterone. Thus, the current study suggests that: (i) the varying pattern of testosterone level and AR status in thyroid tissues of men and women may predispose to the gender specific incidence of thyroid tumors and (ii) miR-124a plays a significant role in determining the AR gene expression pattern and thus, androgen mediated thyroid tumor growth.

Isolated hepatic tuberculosis.

We report a case of carcinoma stomach who had fever of three weeks duration. On evaluation he was detected to have hepatic nodules. It was initially presumed to be due to metastasis. The biopsy of the nodules showed it to be of tubercular in origin. He was treated with antitubercular treatment (ATT) and the fever resolved.

Light and electron microscopic appearances of peripheral nerves from two lepromatous leprosy patients after 12 months of multidrug therapy and their significance.

Biopsies from radial cutaneous nerves of a lepromatous patient and one borderline lepromatous patient treated with 12 doses of multidrug regimen were studied using light and electronmicroscopes. Histopathologically both showed typical lepromatous neuritis. Electronmicroscopic examination showed demyelination, atrophy and degeneration of myelinated axons and nonmyelinated axons and a marked increase in collagen fibrils. Perineurial cells, Schwann cells and endoneurial macrophages contained numerous persisting M. leprae. Almost all the organisms in macrophages were fragmented and could be considered non-viable. A few M. leprae found in Schwann cells showed structure of viable bacilli. It is possible a few dead or dormant organisms may persist for many years in Schwann cells or in fibrous tissue without producing any ill effects, and may cause relapse only in rare instances. Since 12 months of MDT resulted in the clearance of M. leprae in course of time and the reported relapse rates after years were insignificant, implementation of MDT for a year for all MB patients is justified provided surveillance of these patients is ensured. Administration of uniform MDT for 6 months is worth a trial.

Role of endogenous endothelin on coronary reflow after cardioplegic arrest.

OBJECTIVE: Endothelin plays a role in the regulation of basal coronary tone. We hypothesized that low coronary reflow and reduced cardiac function after prolonged ischemia may be due to increased release of endogenous endothelin. METHODS: Using an isolated perfused rat heart, we examined the effect of the addition of various endothelin antagonists during reperfusion after 4 hours of cardioplegic arrest at 4 degrees C. Hearts were freeze-clamped at the end of reperfusion for analysis of high-energy phosphate levels. Results are expressed as the percentages of preischemic values. RESULTS: The addition of bosentan or Ro61-0612 (nonselective endothelin antagonists) resulted in a significant increase in the recovery of coronary flow after 30 minutes of reperfusion (100.9% vs 85.3% [P =.03] and 122.4% vs 83.7% [P <.001], respectively, versus controls). The addition of PD155080 (endothelin A antagonist) had a similar effect (129.5% vs 91.4%, P =.008). BQ788 (endothelin B antagonist) and phosphoramidon (endothelin-converting enzyme inhibitor) had no effect. Myocardial adenosine triphosphate levels were significantly (12.1%) higher after reperfusion with Ro61-0612 (18.1 +/- 0.4 micromol/g vs 16.2 +/- 0.5 micromol/g, P =.01). There was no difference in the recovery of cardiac mechanical function with any of the antagonists studied. CONCLUSION: These results suggest that endogenous endothelin plays a role in low coronary reflow after prolonged cardioplegic arrest but does not impair recovery of myocardial function.

Protection from reperfusion injury after cardiac transplantation by inhibition of adenosine metabolism and nucleotide precursor supply.

BACKGROUND: Adenosine (Ado) triggers numerous protective mechanisms in the heart that may attenuate ischemia-reperfusion injury in cardiac grafts. We aimed to establish whether sustained increase in endogenous Ado production by the combined application of Ado metabolism inhibitors and nucleotide precursors attenuates reperfusion injury in transplanted hearts. METHODS AND RESULTS: Rat hearts were collected after the infusion of St Thomas' Hospital cardioplegic solution, stored at 4 degrees C for 4 hours, and heterotopically transplanted into the abdomen of recipient rats. A solution containing Ado deaminase inhibitor erythro-9(2-hydroxy-3-nonyl)adenine, Ado kinase inhibitor 5'-aminoadenosine, and nucleotide precursors adenine and ribose was administered at the time of reperfusion in the treated group, whereas saline was administered to control animals. After 1 or 24 hours, mechanical function of the transplanted hearts was evaluated in an ex vivo perfusion system followed by the determination of myocardial ATP with related metabolites and measurement of the activity of neutrophil-specific enzyme myeloperoxidase in cardiac homogenates. After 24 hours of reperfusion, maximum left ventricular developed pressure increased from 87.0+/-6.8 mm Hg (mean+/-SEM) in controls to 118.1+/-8.2 mm Hg in the treated group (P<0.05), ATP increased from 11.0+/-0.8 micromol/g dry wt in controls to 15.1+/-1.2 micromol/g dry wt in the treated group (P<0.01), and myeloperoxidase activity decreased from 2.23+/-0.60 U/g wet wt in controls to 0.58+/-0.12 U/g wet wt in the treated group (P<0.001). No differences in cardiac function, ATP, or myeloperoxidase activity were observed between the treated group and controls after 1 hour of reperfusion. CONCLUSIONS: The administration of Ado metabolism inhibitors with nucleotide precursors causes a sustained increase in endogenous Ado production and exerts a potent protective effect against reperfusion injury in transplanted hearts. Improved cardiac function and elevated ATP concentration were accompanied by complete amelioration of neutrophil infiltration in treated hearts, suggesting that reduction in postischemic inflammation could be an important mechanism of this protective effect.

Heat shock protein 70 gene transfection protects mitochondrial and ventricular function against ischemia-reperfusion injury.

BACKGROUND: Upregulation of heat shock protein 70 (HSP70) is beneficial in cardioprotection against ischemia-reperfusion injury, but the mechanism of action is unclear. We studied the role of HSP70 overexpression through gene therapy on mitochondrial function and ventricular recovery in a protocol that mimics clinical donor heart preservation. METHODS AND RESULTS: Hemagglutinating virus of Japan (HVJ)-liposome technique was used to transfect isolated rat hearts via intracoronary infusion of either the HSP70 gene (HSP group, n=16) or no gene (CON group, n=16), which was heterotopically transplanted into recipient rats. Four days after surgery, hearts were either perfused on a Langendorff apparatus for 30 minutes at 37 degrees C (preischemia studies [n=8/group]) or perfused for 30 minutes at 37 degrees C, cardioplegically arrested for 4 hours at 4 degrees C, and reperfused for 30 minutes at 37 degrees C (postischemia studies [n=8/group]). Western blotting and immunohistochemistry confirmed HSP70 upregulation in the HSP group. Postischemic mitochondrial respiratory control indices (RCIs) were significantly better preserved in HSP than in CON hearts: NAD(+)-linked RCI values were 9.54+/-1.1 versus 10.62+/-0.46 before ischemia (NS) but 7.98+/-0.69 versus 1.28+/-0.15 after ischemia (P<0.05), and FAD-linked RCI values were 6.87+/-0.88 versus 6.73+/-0.93 before ischemia (NS) but 4.26+/-0.41 versus 1.34+/-0.13 after ischemia (P<0.05). Postischemic recovery of mechanical function was greater in HSP than in CON hearts: left ventricular developed pressure recovery was 72.4+/-6.4% versus 59.7+/-5.3% (P<0.05), maximum dP/dt recovery was 77.9+/-6.6% versus 52.3+/-5.2% (P<0.05), and minimum dP/dt recovery was 72.4+/-7.2% versus 54.8+/-6.9% (P<0.05). Creatine kinase release in coronary effluent after reperfusion was 0.20+/-0.04 versus 0.34+/-0.06 IU. min(-1). g wet wt(-1) (P<0.05) in HSP versus in CON hearts. CONCLUSIONS: HSP70 upregulation protects mitochondrial function after ischemia-reperfusion injury; this was associated with improved preservation of ventricular function. Protection of mitochondrial function may be important in the development of future cardioprotective strategies.

Efficacy of adenoviral gene transfer with manganese superoxide dismutase and endothelial nitric oxide synthase in reducing ischemia and reperfusion injury.

OBJECTIVE: Both superoxide dismutase (SOD), a free radical scavenger, and nitric oxide (NO), a vasodilator with anti-inflammatory properties, have been shown to protect the myocardium from reperfusion injury. They are known to interact in vivo, the influence of which on myocardial protection has not been studied. METHODS: Four groups of rats (n=7, per group) were subjected to experimental infarction following injections into the anterior wall of the left ventricle with adenoviral vector encoding beta-galactosidase (group A), eNOS (group B), Mn-SOD (group C) and both eNOS and MnSOD (group D). Hearts were assessed for protein expression and size of infarction. RESULTS: Efficiency of gene up regulation was confirmed by immunostaining for eNOS and Mn-SOD, and X-gal staining for beta-gal respectively. In B and D, overexpression of eNOS was demonstrated in cardiac myocytes in addition to that in the endothelium, while in C and D, Mn-SOD was overexpressed in mainly cardiomyocytes. Infarct size was 49.7+/-4.8% in A, and was significantly reduced in the other groups (29.8+/-2.7%, 21.8+/-2.5% and 24.9+/-2.4% in B, C and D respectively). CONCLUSION: Adenoviral gene transfer of Mn-SOD was superior to eNOS in reducing the extent of in vivo ischemia-reperfusion injury in the rat heart in our model. The effect of combined application of Mn-SOD and eNOS was not different from their individual effect.

Pyruvate/dichloroacetate supply during reperfusion accelerates recovery of cardiac energetics and improves mechanical function following cardioplegic arrest.

OBJECTIVES: Cardioplegic arrest during cardiac surgery induces severe abnormalities of the pyruvate metabolism, which may affect functional recovery of the heart. We aimed to evaluate the effect of pyruvate and dichloroacetate administration during reperfusion on recovery of mechanical function and energy metabolism in the heart subjected to prolonged cardioplegic arrest. METHODS: Four groups of rat hearts perfused in working mode were subjected to cardioplegic arrest (St. Thomas' No. 1), 4 h of ischaemia at 8 degrees C and reperfusion with either Krebs buffer alone (C) or with 2.8 mM pyruvate (P), with 1 mM dichloroacetate (D), or with a combination of both (PD). Mechanical function was recorded before cardioplegic arrest and at the end of experiments. In groups C and PD, additional experiments were performed using (31)P nuclear magnetic resonance spectroscopy in non-working Langendorff mode to evaluate cardiac high-energy phosphate concentration changes throughout the experiment. RESULTS: Improved recovery of cardiac output (% of the preischaemic value+/-SEM, n=9-12) was observed in all three treated groups (65.7+/-4.3, 59.5+/-5.2 and 59.5+/-5.3% in PD, P and D, respectively) as compared with C (42.2+/-4.6%; P<0.05). Recovery of coronary flow was improved from 66.4+/-3.8 in C to 94.9+/-8.6% in PD (P<0.05). The phosphocreatine recovery rate in the first minutes of reperfusion was increased from 9.9+/-1.5 in C to 31.5+/-4.3 micromol/min per g dry wt in PD (P<0.001). No differences were observed in ATP or phosphocreatine concentrations at the end of experiment. CONCLUSIONS: The administration of pyruvate and dichloroacetate improves the recovery of mechanical function following hypothermic ischaemia. Accelerated restoration of the energy equilibrium in the initial phase of reperfusion may underlie the metabolic mechanism of this effect.

Gene therapy for myocardial protection: transfection of donor hearts with heat shock protein 70 gene protects cardiac function against ischemia-reperfusion injury.

BACKGROUND: Heat shock protein 70 (HSP70) gene transfection has been shown to enhance myocardial tolerance after normothermic ischemia-reperfusion. We investigated the effect of HSP70 gene transfection on mechanical and endothelial function in a protocol mimicking clinical heart preservation. METHODS AND RESULTS: Rat hearts were infused ex vivo with Hemagglutinating Virus of Japan-liposome complex containing HSP70 gene (HSP, n=8) or no gene (CON, n=8), and heterotopically transplanted into recipient rats. Four days after surgery, transfected hearts were perfused on a Langendorff apparatus for 45 minutes, arrested with St Thomas' No. 1 cardioplegia for 4 hours at 4 degrees C, and reperfused for 1 hour. Mechanical and endothelial function was studied before and after ischemia. Creatine kinase was measured in reperfusion effluent. Hearts underwent Western blotting and immunohistochemistry to confirm HSP70 overexpression. Postischemic recovery of mechanical function (% preischemic+/-SEM) was greater in HSP versus CON: Left ventricular developed pressure recovery was 76.7+/-3.9% versus 60. 5+/-3.1% (P:<0.05); dP/dtmax recovery was 79.4+/-4.9% versus 56. 2+/-3.2% (P:<0.05); dP/dtmin recovery was 74.8+/-4.6% versus 57. 3+/-3.6% (P:<0.05). Creatine kinase release was attenuated in HSP versus CON: 0.22+/-0.02 versus 0.32+/-0.04 IU/min/g wet wt. (P:<0. 05). Recovery of coronary flow was greater in HSP versus CON: 76. 5+/-3.8% versus 59.2+/-3.2% (P:<0.05). Recovery of coronary response to 5-hydroxytryptamine (5 x 10(-)(5) mol/L) was 55.6+/-4.7% versus 23. 9+/-3.2% (P:<0.05); recovery of coronary response to glyceryltrinitrate (15 mg/L) was not different between HSP and CON: 87.4+/-6.9% versus 84.3+/-5.8% (NS). CONCLUSIONS: In a clinically relevant donor heart preservation protocol, HSP70 gene transfection protects both mechanical and endothelial function.

Development of a novel method for cell transplantation through the coronary artery.

BACKGROUND: Cell transplantation is a promising strategy to treat end-stage heart failure. At present, a popular method to deliver cells into the heart is direct intramuscular injection. This method, however, may not be efficient in spreading cells globally into the myocardium. We have developed a novel method for cell transplantation using intracoronary infusion. METHODS AND RESULTS: An L6 rat skeletal muscle cell line expressing ss-galactosidase (ss-gal) was generated by gene transfection and clonal selection. These cells (10(6) in 1 mL medium) were infused into explanted rat hearts through the coronary artery, followed by heterotopic heart transplantation into the abdomen of recipients. Control hearts were infused with cell-free medium. According to ss-gal activity measurements, approximately 5 x 10(5) grafted cells per heart existed on day 3, increasing to 5 x 10(6) on day 28 in the cell-transplanted hearts. At day 28, discrete loci positively stained for ss-gal were observed throughout the cardiac layers of both left and right coronary territories. Some of them differentiated into ss-gal-positive multinucleated myotubes that aligned with the cardiac fiber axis and integrated into the native myocardium, whereas others formed colonies consisting of undifferentiated myoblasts. Connexin 43, a cardiac gap junction protein, was expressed between grafted cells and native cardiomyocytes. No reduction in cardiac function was observed in a Langendorff perfusion system. CONCLUSIONS: We have developed a unique method for efficient cell transplantation based on intracoronary infusion. This method, potentially applicable in the clinical setting during cardiac surgery, could be useful to globally supply cells to the heart.

Effects of heat stress on metabolism of high-energy phosphates. Comparison of normothermic and hypothermic ischemia.

BACKGROUND: Alterations in metabolic pathways may contribute to the cardioprotective effects of heat stress (HS). We investigated the effects of HS on ATP and phosphocreatine (PCr) levels in the ischemic rat myocardium, after both normothermic and hypothermic ischemia. METHODS: Two protocols were used: (1) normothermic ischemia (20 min at 37 degrees C) with no myocardial protection (n=6 HS; n=6 control); (2) hypothermic ischemia (4 hrs at 4 degrees C) after cardioplegic arrest (n=6 HS; n=6 control). ATP and PCr levels in the heart were measured using 31P nuclear magnetic resonance spectroscopy. RESULTS: At the end of normothermic ischemia, ATP levels were better maintained in HS hearts (C vs HS: 4.51+/-0.66 vs 7.81+/-1.06 micromol/g dry wt+/-SEM, p=0.04). A trend for higher ATP content in HS hearts was observed after 40 min of reperfusion (C vs HS: 11.7+/-1.5 vs 16.9+/-2.0 micromol/g dry wt+/-SEM, p=0.09). PCr content was also higher at the end of 40 minutes of reperfusion in HS hearts (C vs HS: 46.4+/-2.9 vs 56.9+/-3.0 micromol/g dry wt+/-SEM, p=0.03). After prolonged hypothermic ischemia under cardioplegic arrest, heat stress again led to better preservation of ATP levels at the end of ischemia (C vs HS: 5.71+/-0.88 vs 9.23+/-1.38 micromol/g dry wt+/-SEM, p=0.05) and after 40 minutes of reperfusion (C vs HS: 16.8+/-1.4 vs 24.6+/-2.8 micromol/g dry wt+/-SEM, p=0.03). PCr levels were also better maintained at the end of ischemia (C vs HS: 4.87+/-0.77 vs 12.4+/-3.0 micromol/g dry wt+/-SEM, p=0.03) and after 40 minutes of reperfusion in HS hearts (C vs HS: 55.1+/-7.0.vs 79.8+/-7.3 micromol/g dry wt+/-SEM, p=0.03). CONCLUSIONS: Heat stress induces changes in the energy profile of the heart which results in better preservation of ATP and phosphocreatine levels. These changes could be observed after brief normothermic ischemia and also after prolonged hypothermic ischemia under cardioplegic arrest, mimicking conditions of preservation for cardiac transplantation.

Influence of heat stress on myocardial metabolism and functional recovery after cardioplegic arrest: a 31P N.M.R study.

OBJECTIVE: Heat stress and induction of heat shock proteins confer protection against myocardial ischemia-reperfusion injury; however the precise mechanisms of this effect remain unknown. We investigated the influence of heat stress on metabolic and functional recovery after cardioplegic arrest, in a protocol mimicking clinical donor heart preservation. METHODS: Langendorff perfused rat hearts in control group (C, n = 6) and heat stressed (24 h prior to experiment) group (HS, n = 6) were subjected to 4 h of ischemia at 4 degrees C following cardioplegic arrest (St. Thomas' No. 1). 31P nuclear magnetic resonance spectroscopy was used to follow changes in ATP, phosphocreatine and inorganic phosphate concentrations during the pre-ischemic, ischemic and reperfusion periods. Myocardial adenine nucleotide levels in hearts at the end of experiments and purine catabolite release in coronary effluent during reperfusion, were evaluated using high performance liquid chromatography. Mechanical function in the pre-ischemic and reperfusion periods was evaluated using an intraventricular balloon. Western immunoblotting was used to quantitate HSP70 expression. RESULTS: Although baseline concentrations of ATP and phosphocreatine were similar in C and HS groups, the rate of high-energy phosphate depletion was attenuated during the early phase of ischemia in HS groups. On reperfusion, recovery of ATP was 10-20% greater in HS versus C groups; phosphocreatine levels also recovered better in the HS group, transiently reaching levels 40% higher in HS versus C groups. The concentrations of adenine nucleotides in hearts were significantly higher in the HS versus C groups. These changes were associated with an attenuation of total purine catabolite release in the coronary effluent in HS versus C groups. A significant improvement in relative recovery of developed pressure was shown in HS versus C groups in the post-ischemic periods. CONCLUSIONS: Heat stress causes beneficial changes in high-energy phosphate metabolism in the rat heart subjected to cardioplegic arrest and ischemia. Improved mechanical recovery in HS versus C groups was associated with a decreased rate of high-energy phosphate depletion and increased recovery of ATP and phosphocreatine levels during reperfusion. Changes in energy metabolism may play a role in the mechanism of cardioprotection by heat stress during prolonged hypothermic cardiac arrest. rights reserved.

Influence of ageing on functional recovery and guanine nucleotide levels of the heart following cold cardioplegic arrest.

OBJECTIVE: The effect of age on metabolism and mechanical recovery of the heart after cardioplegic arrest is important, but remains a relatively unexplored subject. In this study, functional recovery and nucleotide levels were compared in the heart at different ages subjected to prolonged hypothermic cardioplegic arrest. METHODS: Three different age groups of rats: 1 (A); 4 (B); and 16 months (C) were perfused in working mode and subjected to cardioplegic arrest (St. Thomas' No. 1) and ischemia for 4 h at 4 degrees C, followed by reperfusion for 35 min. Cardiac function (cardiac output and aortic pressure) was recorded before and after ischemia. Another series of hearts in all three age groups underwent 5 min of normoxic perfusion to obtain pre-ischemic baseline metabolite concentrations. Hearts were freeze-clamped at the end of each experiment and used for determination of nucleotide and creatine metabolites by HPLC. RESULTS: The post-ischemic recovery (% of the pre-ischemic value) of the cardiac power was 48.9 +/- 7.8% for group A, which was significantly higher than the functional recovery of group B (24.1 +/- 3.5%) or C (21.4 +/- 4.7%, P < 0.05, respectively). There was no difference in ATP or the total adenine nucleotide or creatine metabolite concentrations between the three age groups. In contrast, both GTP and the total guanine nucleotide concentration was highest in A (P < 0.05). Total guanylate pool was 1.52 +/- 0.10 1 micromol/g dry wt. in A, as compared to B (1.05 +/- 0.04) or C (1.12 +/- 0.04). NAD was significantly higher in B (4.1 +/- 0.1. P < 0.05), when compared to A (3.6 +/- 0.1) and C (3.8 +/- 0.1). CONCLUSION: Best post-ischemic functional recovery after cardioplegic arrest was observed in the 1-month-old hearts (A) and was associated with highest guanine nucleotide concentration; preservation of guanine nucleotide pool in the youngest hearts may be an important mechanism for improved cardioprotection due to the important role of GTP in signalling pathways.