Nutrient content and stoichiometry of pelagic Sargassum reflects increasing nitrogen availability in the Atlantic Basin.

The pelagic brown macroalgae Sargassum spp. have grown for centuries in oligotrophic waters of the North Atlantic Ocean supported by natural nutrient sources, such as excretions from associated fishes and invertebrates, upwelling, and N2 fixation. Using a unique historical baseline, we show that since the 1980s the tissue %N of Sargassum spp. has increased by 35%, while %P has decreased by 44%, resulting in a 111% increase in the N:P ratio (13:1 to 28:1) and increased P limitation. The highest %N and δ15N values occurred in coastal waters influenced by N-rich terrestrial runoff, while lower C:N and C:P ratios occurred in winter and spring during peak river discharges. These findings suggest that increased N availability is supporting blooms of Sargassum and turning a critical nursery habitat into harmful algal blooms with catastrophic impacts on coastal ecosystems, economies, and human health.

Intermittent Access to Ethanol Induces Escalated Alcohol Consumption in Primates.

BACKGROUND: Escalation of voluntary alcohol drinking is characteristic of alcohol addiction and can be induced in rodents using intermittent access to alcohol. This model has been used to evaluate candidate therapeutics, but key systems involved in the transition into alcohol addiction, such as CRF, differ in their organization between rodents and primates. We examined the ability of an intermittent access schedule to induce escalation of voluntary alcohol drinking in non-human primates and used this model to assess the role of corticotropin releasing hormone (CRF) signaling in this process. METHODS: Four young adult male rhesus macaques were given access to an 8.4% alcohol solution every other weekday (EOD; M, W, F), while four other young adult males were given the same solution every weekday (ED; M-F). Subjects were then administered a CRF1 antagonist, antalarmin. RESULTS: EOD increased alcohol intake by up to 50% over baseline, with a more pronounced increase immediately following reintroduction of alcohol. For the morning/daytime sessions, EOD subjects increased their consumption by 83% over baseline. Differences between ED and EOD schedules emerged quickly, and EOD-induced escalation resulted in pharmacologically active BAC's. EOD-induced alcohol consumption was insensitive to CRFR1 blockade by antalarmin, but subjects with high CSF levels of CRF were more responsive. CONCLUSIONS: Similar to what has been observed in rodents, intermittent access results in an escalation of voluntary alcohol drinking in non-human primates. In contrast to findings in rats, recruitment of the CRF system does not seem to be involved in the escalated alcohol drinking observed under these conditions, though individual differences in CRF system activity may play a role.

Early experience and sex interact to influence limbic-hypothalamic-pituitary-adrenal-axis function after acute alcohol administration in rhesus macaques (Macaca mulatta).

BACKGROUND: Studies in rodents demonstrate sex differences in neuroendocrine stress axis activity after treatment with alcohol. In abstinent alcoholics, atypical depressives, and individuals with posttraumatic stress disorder, limbic-hypothalamic-pituitary-adrenal (LHPA)-axis activity is often blunted; among females in these patient populations, however, resistance to glucocorticoid feedback and increased pituitary reactivity is observed. Early parental loss is a major life stressor and is a risk factor for both affective disturbances and LHPA-axis abnormalities later in life. We wanted to determine whether sex and early life parental absence would interact to influence alcohol-induced alterations in LHPA-axis activity after exposure to ethanol in macaques. METHODS: Animals were reared with their mothers in social groups (MR, n = 94) or without adults in peer-only groups (PR, n = 79). At 5 years of age, they received an intravenous infusion of alcohol (2-2.2 g/kg), and the effects of alcohol, sex, and rearing condition on ACTH and cortisol levels were analyzed by ANOVA. RESULTS: Peer-reared females had higher ACTH levels than did PR males, MR females, and MR males after alcohol infusion. Alcohol-induced cortisol levels were not affected by sex and rearing condition. CONCLUSIONS: These findings suggest that there are sex differences in glucocorticoid negative feedback, pituitary responsivity, or release of ACTH secretagogues among individuals exposed to early life stress and emphasize the importance of considering sex effects when studying LHPA-axis dysregulation in alcoholism and other stress-related neuropsychiatric disorders.

Donor brain death reduces survival after transplantation in rat livers preserved for 20 hr.

BACKGROUND: Eighty percent of donor organs come from donors who have suffered brain trauma (brain-dead donors). This unphysiological state alters the hemodynamic and hormonal status of the organ donor. This can cause organ injury, which has been suggested to alter the immunological or inflammatory status of the organ after transplantation, and may lead to increased sensitivity of the organ to preservation/transplantation injury. In this study we asked the question: does brain death cause injury to the liver that decreases successful liver preservation? METHODS: The rat liver transplant model was used to compare survival in rats receiving a liver from a brain-dead donor versus a non-brain-dead donor. Brain death was induced by inflation of a cranially placed balloon catheter. The rats were maintained normotensive with fluid infusion for 6 hr. The livers were flushed with University of Wisconsin (UW) solution and immediately transplanted or cold stored for 20 hr before transplantation. RESULTS: Recipient survival with immediately transplanted livers or those stored for 20 hr was 100% with livers from non-brain-dead donors. However, survival decreased when livers were procured from brain-dead donors. Survival was 75% (6/8) when storage time was 0 hr and 20% (2/10) when the liver was cold stored for 20 hr before transplantation. CONCLUSION: This study shows that brain death induces alterations in the donor liver that make it more sensitive to preservation/reperfusion injury than livers from donors without brain death. The mechanism of injury to the liver caused by brain death is not known. Because most livers used clinically for transplantation come from brain-dead donors, it is possible that poor function of these livers is due to the intrinsic condition of the donor organ, more than the quality of the preservation. Methods to treat the brain-dead donor to improve the quality of the liver may be needed to allow better preservation of the organ and to give better outcome after liver transplantation.

Design and synthesis of inhibitors of adenosine and AMP deaminases.

Nucleosides and nucleotides which are able to undergo covalent hydration in the aglycone ring system are potential inhibitors of the enzymes adenosine deaminase (ADA) and AMP deaminase, respectively. Calculations of the enthalpy of covalent hydration and of enzyme binding energy have been used to design new inhibitors of ADA. The ribosyl triazolotriazine 16, which was synthesized as a result of these calculations, exists predominantly as the covalent hydrate 18 in water and is a potent inhibitor of mammalian ADA (IC(50) 50 nM).

Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice.

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

Salivary prolactin as a marker for central serotonin turnover.

Central nervous system (CNS) serotonin deficits have been linked to many pathological behaviors in both human and nonhuman primates. The plasma prolactin response to fenfluramine has been widely used to assess CNS serotonin functioning in humans. Prolactin is also found as an integrated measure in saliva. We hypothesized that salivary prolactin concentrations would correlate positively with cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) in rhesus monkeys. Twenty-seven adult male and female rhesus macaques (Macaca mulatta) were sampled for concurrent saliva, blood, and CSF. Saliva and blood serum were assayed for prolactin concentrations, and CSF was assayed for 5-HIAA, homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG). Salivary prolactin concentrations were positively correlated with CSF 5-HIAA concentrations. No other relationships between any of the measures, including that between salivary prolactin and serum prolactin, were found to be statistically significant. These findings suggest the possibility of using salivary prolactin concentrations as an index of CNS serotonin turnover in humans.

The design and synthesis of inhibitors of adenosine 5'-monophosphate deaminase.

Carbocylic coformycin (4) is a potent herbicide whose primary mode of action involves inhibition of adenosine 5'-monophosphate deaminase (AMPDA) following phosphorylation of the 5'-hydroxyl group in vivo. The search for more stable and accessible structures led to the synthesis of carbocyclic nebularine (8) and deaminoformycin (10). The latter compound is a good herbicide and its corresponding 5'-monophosphate 14 is a strong inhibitor of plant AMPDA (IC50 100 nM).

Extended preservation and effect of nitric oxide production in liver transplantation.

Liver transplantation (Ltx) has become a routine procedure for patients with end-stage liver disease. Despite ongoing progress on short- and long-term graft survival, primary dysfunction (PDF) remains a major problem. PDF is significantly associated with the duration of cold ischemia- and, possibly, with reperfusion-related injury. Nitric oxide (NO) has many physiological functions and plays an important role in modulating tissue injury. However, the mechanism of NO action in ischemia/reperfusion injury after Ltx is thus far unknown. In this study we investigated the role of inducable NO synthase (iNOS) in the liver after preservation with UW solution using the orthotopic Ltx model in the rat. Male Brown Norway rats were used for the Ltx procedure. After donor hepatectomy, livers were stored on ice-cold UW solution for 24 or 40 h and subsequently transplanted. A control group consisted of rats with Ltx after less than 1 h storage. Post-transplant blood samples were taken at 48 h to determine standard parameters for liver injury (aspartate transaminase, lactate dehydrogenase). Liver biopsies were obtained for detection of expression of iNOS (western blot) 24 and 48 h post-transplant. We observed that a preservation time of 24 h in UW solution presents no problem for graft survival after Ltx in rats with some brain function and in healthy animals. After 40 h preservation, liver damage is obvious and graft survival reduced, indicating the limits of cold storage may be within reach. With longer preservation times, more NOs was detected in liver tissue. This finding suggests that NO has a role in ischemia/reperfusion-related injury. Current intervention with NOS inhibitors will reveal whether NO has a negative or a positive effect on graft survival after Ltx.

Optimal pH for simple cold storage or machine perfusion of dog kidneys with UW solution.

Metabolic suppression by temperature is a key to successful organ preservation. Additional methods for inducing metabolic suppression may further improve organ preservation. Extracellular acidosis has been shown to suppress warm anoxic injury to various isolated cells. Acidosis may suppress enzymes with a pH optimum at the pH of the cytosol (pH 7.3). In this study, the combination of hypothermia and acidosis was used to determine if it would improve renal preservation. Dog kidneys were cold-stored (CS) for 48 h in University of Wisconsin (UW) solution with the pH adjusted to 6.4, 6.8, 7.4, or 7.8. Kidneys were also machine-perfused (MP) for 3 days with the gluconate perfusion solution (Belzer's machine perfusion solution, MPS) at pHs similar to those tested for CS. Renal function (serum creatinine, SCr) and survival were recorded in immediate contralateral nephrectomized recipients. On the basis of maximum SCr values, kidneys preserved by CS or MP were best preserved at pHs of 7.4 or 7.8. At a pH of 6.8, SCr values were elevated and returned to normal at a slower rate than in those preserved at higher pHs. This study shows that acidosis is not cytoprotective to cold-stored dog kidneys and causes preservation/reperfusion injury.

Adenosine-5'-phosphate deaminase. A novel herbicide target.

The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool.

Cardiovascular risk factors in commercial flight aircrew officers compared with those in the general population.

Cardiovascular disease is the most common reason for loss of license among commercial flight pilots. This study was done to explore cardiovascular risk factors among aircrew officers. The study group consisted of 113 male commercial flight aircrew officers (aviators), aged thirty-five to forty-four years (mean: 38.8 years) who participated in the compulsory health screening. Men investigated at the Health Screening Centre, Malmö, were used as the reference group. Group 1, for ECG, (n 771), aged thirty-eight to forty-four years (mean: 42.1). Group 2, for height, weight, body mass index (BMI) (weight kg/height m2), blood pressure, serum cholesterol (total), and smoking habits (n 5005), aged thirty-five to forty-four years (mean: 39.2). The aviators did not differ from the reference population in regard to height, weight, BMI, diastolic blood pressure, or smoking habits. However, the incidences of electrocardiographic left ventricular hypertrophy, increased systolic blood pressure, and the level of cholesterol were significantly higher in the aviators when compared with the controls. Aircrew members may primarily be selected by criteria that differ from the male population in general. Excessive environmental stress, ie, shift work, jet lag, fatigue, as well as dietary factors, may also contribute to anomalies in the group. The clinical consequences of these anomalies for the aviators should be further evaluated, for they are important both for the aviators and for flying safety.

Excessive mortality in young free-ranging male nonhuman primates with low cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations.

BACKGROUND: The purpose of this study was to assess the impact of central serotonin turnover rate on survival to adulthood among nonhuman primates living in a large, free-ranging colony. METHODS: The rate of mortality was ascertained over a 4-year period after obtaining blood and cisternal cerebrospinal fluid (CSF) samples from 49 free-ranging, 2-year-old prepubertal male rhesus monkeys. Cerebrospinal fluid was assayed for 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine, 3-methoxy-4-hydroxyphenylgycol, and homovanillic acid. Blood plasma was assayed for adrenocorticotropic hormone, cortisol, and testosterone. Following the sampling of body fluids, records of scars and wounds and aggressive encounters were used to rank the subjects from low to high in aggressiveness. Direct observations of aggressive behavior were collected from 27 of the subjects over a 3-month period. RESULTS: Four years later, 6 of the 49 subjects were known to be dead and an additional 5 had been missing for more than 2 years and were presumed dead. The CSF 5-HIAA concentrations were predictive of which subjects died, with 46% of the subjects with low CSF 5-HIAA concentrations dead or presumed dead. None of the subjects from the highest CSF 5-HIAA concentration quartile were dead or missing. Indeed, 91% of the dead subjects came from the 2 lowest quartiles of CSF 5-HIAA concentrations. Direct observations of aggressive behavior showed that dead or missing subjects had initiated escalated aggression, a measure of unrestrained aggression that has a high probability of trauma or injury, at a higher rate than subjects that were known to be alive. The cause of death could be ascertained for 6 of the 11 missing subjects. The 4 subjects that were known to die as a consequence of aggressive encounters came from the lowest quartile of CSF 5-HIAA concentrations and had been rated as more aggressive during their initial capture. Subjects captured more than once possessed lower CSF 5-HIAA concentrations, were rated as more aggressive, and were more likely to suffer early death than those captured only once. CONCLUSION: These findings suggest that low CSF 5-HIAA concentrations quantified early in life is a powerful biological predictor of future excessive aggression, risk taking, and premature death among nonhuman primate males.

Donor nutritional status--a determinant of liver preservation injury.

In liver transplantation, the quality of the liver is determined by a number of factors including donor nutritional status. Livers from fasted donors appear to tolerate long-term preservation better than livers from fed donors. In this study we repeated earlier results and obtained 31% (4/13) survival after 40-hr preservation of livers from fed donor Brown Norway rats and 67% (8/12) survivors with donor livers from 4-day-fasted rats (P = 0.154). The explanation for this improvement is not known but may be due to inactivation of Kupffer cells due to nutritional depletion of the liver. Kupffer cell activation has been one explanation advanced to explain how cold storage injuries livers during reperfusion (transplantation). In this study, we have measured how donor fasting affects Kupffer cell function (phagocytosis of colloidal carbon) after preservation of the rat liver. In addition, we measured how enhancing liver glycogen by feeding glucose to the rat donors affected outcome and liver functions tested by isolated perfusion after 24- and 40-hr cold storage of the liver. Preservation did not cause inactivation or activation of Kupffer cell phagocytosis of colloidal carbon. In livers with 0-hr preservation, colloidal carbon uptake was 3.1 +/- 0.2 mg/g/hr, after 40-hr preservation uptake was 3.8 mg/g/hr (P < 0.05 vs. 0 hr) (fed) and 2.7 +/- 0.3 mg/g/hr (fasted, P, 0.05 vs. 0-hr and 40-hr-fed). Thus, the improved survival obtained with livers from fasted donors does not appear related to inactivation of Kupffer cell phagocytosis. Although livers from fasted donors showed improved survival, there was extensive hepatocellular injury as indicated by large LDH release from the livers after 40-hr cold storage as tested by isolated perfusion. LDH released into the perfusate increased from 35.8 +/- 10.1 U/L (fed, 40-hr CS) to 301 +/- 65 U/L (fasted, 40-hr CS) after 1-hr reperfusion. AST release showed a similar pattern and bile production was suppressed more in livers from fasted donors than fed donors. Feeding rats glucose elevated liver glycogen and significantly reduced hepatocellular injury as measured by LDH release and AST release in the isolated perfused liver after 40-hr cold storage. Feeding rats glucose (40% in drinking water for 4 days) also improved survival: fed+glucose = 85% survival versus 31% survival with no glucose and fasted+glucose = 92% survival versus 67% survival with no glucose. These results show that both extensive donor fasting and glucose feeding enhanced outcome in orthotopic liver transplantation. This dilemma (both fasting and feeding improved survival) are discussed in terms of how the interactions between Kupffer cells and hepatocytes affect liver viability. Donor fasting is probably impractical clinically as a method to improve the donor liver, but elevating liver glycogen by glucose supplementation is possible and may lead to improved preservation and outcome in liver transplantation.

Ten year cerebrovascular morbidity and mortality in 68 year old men with asymptomatic carotid stenosis.

OBJECTIVE: To study the natural course of carotid artery stenosis detected by ultrasonography. DESIGN: Prospective cohort study. Baseline examination in 1982-3 included ultrasound examination of carotid arteries, measurement of ankle-brachial blood pressure index, and detection of atrial fibrillation by 24 hour ambulatory electrocardiography. SETTING: Malmö, a city in southern Sweden with 230,000 inhabitants. SUBJECTS: 470 men aged 68 years randomly selected from the population. MAIN OUTCOME MEASURES: Incidence of stroke and transient ischaemic attack and all cause mortality during 10 years of follow up in relation to carotid stenosis, leg artery disease (ankle-brachial blood pressure index below 0.9), and atrial fibrillation. RESULTS: Fifty men had a stroke; six of these were haemorrhagic. Another 11 had a transient ischaemic attack. Eighteen of the men with carotid stenosis (21.6 events/1000 person years) and 43 of the men with normal carotid arteries (14.8 events/1000 person years) had a stroke or transient ischaemic attack (P = 0.188). Men with atrial fibrillation had an increased rate of cerebrovascular events (36.7/1000 person years (P = 0.048). The highest rate was found in men with asymptomatic disease of the leg arteries (38.6/1000 person years) (P < 0.001). The increased risk of stroke or transient ischaemic attack in this group remained after multivariate analysis (relative risk 2.0; 95% confidence interval 1.1 to 3.7). CONCLUSIONS: In this cohort carotid stenosis was not associated with an increased risk of stroke. Part of this lack of association was explained by the high mortality from ischaemic heart disease in men with severe stenosis. Twenty seven of the 61 cerebrovascular events, however, occurred in men who had normal carotid arteries, normal ankle pressure, and no atrial fibrillation.

Plethysmographic pulse wave amplitude and future leg arteriosclerosis.

A low pulse wave amplitude during calf plethysmography at 55 years of age was previously found to be associated with an increased mortality and incidence of myocardial infarction. In order to test the hypothesis that a low pulse wave amplitude is associated with an increased risk of future leg atherosclerosis as well, we have studied the relationship between a low ankle-brachial pressure index (ABPI; < 0.9) at 68 years of age and the pulse wave amplitude at 55 years of age in that same cohort. The prevalence of a low pulse wave amplitude (< or = 5 mm; lowest quintile) among men with a low ABPI (42%) was more than twice as high as it was among men who had a normal ABPI (19%) (P < 0.001). No association was found between a low ABPI and the plethysmographically recorded leg blood flow at 55 years of age. A low pulse wave amplitude might reflect early symptom-free arteriosclerosis, or age-dependent non-arteriosclerotic loss of vessel wall elasticity. The relationship between a low pulse wave amplitude and a low ABPI remained when controlling for smoking, hypertension and hyperlipidaemia. It is concluded that pulse wave measurement by plethysmography contributes information to improve leg atherosclerotic risk assessment in individuals exposed to known risk factors.

Kupffer cells depress hepatocyte protein synthesis on cold storage of the rat liver.

The causes of liver failure after transplantation are multifactorial. An understanding of the mechanisms of injury to the liver could help to define methods to improve preservation and transplantation. We measured protein synthesis by 3H-leucine incorporation into acid precipitable protein in rat liver tissue slices, isolated hepatocytes, and isolated perfused liver (IPL) after cold storage for 24 or 48 hr in University of Wisconsin (UW) solution. Some rats were pretreated with dexamethasone prior to liver harvest. Protein synthesis was depressed in all in vitro models after 24 hr storage. The percent decrease was greater in tissue slices and IPL (about 70% decrease relative to fresh livers) than in isolated hepatocytes (about 30% decrease). Dexamethasone pretreatment improved protein synthesis significantly after 24 hr preservation in tissue slices and in IPL, but had no significant effect on protein synthesis in isolated hepatocytes. The greater loss of protein synthesis in tissue slices and IPL compared with that in isolated hepatocytes was considered in relation to the presence of Kupffer cells in the former systems and lack of Kupffer cells in the isolated cell suspensions. Kupffer cells generate cytotoxins that could cause injury to metabolically depressed hepatocytes or endothelial cells. Dexamethasone has been shown to modulate Kupffer cell inhibition of hepatocyte functions. The results suggest that preservation damage to hepatocytes sensitizes them to further damage on reperfusion by Kupffer cell-generated agents.

Low pulse-wave amplitude during reactive leg hyperaemia: an independent, early marker for ischaemic heart disease and death. Results from the 21-year follow-up of the prospective cohort study 'Men born in 1914', Malmö, Sweden.

OBJECTIVE: To study the incidence of myocardial infarction, all-cause mortality and mortality from ischaemic heart disease in relation to arterial leg blood flow determined by venous occlusion plethysmography of the calf. DESIGN: A prospective cohort study 'Men born in 1914'. SETTING: Malmö, a city in southern Sweden with 256,000 inhabitants, and a single referral hospital. SUBJECTS: Six-hundred and thirty-six 55-year-old men, randomly selected from the general population. None of them had signs or symptoms of leg artery disease. MAIN OUTCOME MEASURES: All-cause mortality, morbidity and mortality from ischaemic heart disease during 21 years of follow-up following the initial examination in 1968. RESULTS: A low pulse-wave amplitude (i.e. < 5 mm) during reactive hyperaemia was, independently of other known arteriosclerotic risk factors, associated with a higher cardiac event rate of 37.1% (relative risk: 2.2; 95% CI: 1.3-3.6) and a higher all-cause mortality rate of 62.9% (relative risk: 1.7; 95% CI: 1.2-2.4) during 21 years of follow-up. No other plethysmographically recorded variable was associated with an increased mortality and cardiac event rate. CONCLUSIONS: The plethysmographically recorded pulse-wave amplitude during reactive hyperaemia can be used as an early independent marker to identify individuals at risk of developing ischaemic heart disease and death at an early age.