romanian journal of internal medicine

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TEODORA MOCAN, LUCIA AGOŞTON-COLDEA, L.D. RUSU, RALUCA PAIS, ... ELIZA GRAMADĂ, CRISTIANA POPP, FLORICA STĂNICEANU, ŞTEFANA ...

ROMANIAN JOURNAL OF INTERNAL MEDICINE Volume 46

No. 4, 2008

CONTENTS REVIEWS H. BĂLAN, Chronotherapy of Hypertension: When Can Be As Important As With What ........................................................ ADINA STOICA, CARMEN GINGHINĂ, Cardiovascular Risk in Patients with Peripheral Vascular Diseases ...................... ADRIANA GABRIELA FILIP, SIMONA CLICHICI, DOINA DAICOVICIU, DIANA OLTEANU, ADRIANA MUREŞAN, SIMINA DREVE, Photodynamic Therapy – Indications and Limits in Malignant Tumors Treatment ...... NICOLETA MITROI, MARIA MOŢA, Nutrigenomics/Nutrigenetics ......................................................................................

269 275 285 295

ORIGINAL ARTICLES RALUCA MIHĂILESCU, VICTORIA ARAMĂ, SIMONA PARASCHIV, A. STREINU-CERCEL, D. OŢELEA, DANIELA MUNTEANU, MIHAELA IOSIPENCO, CARMEN CHIOTAN, OTILIA ELISABETA BENEA, MARIANA MĂRDĂRESCU, MIHAELA RĂDULESCU, ADRIANA HRISTEA, RODICA UNGURIANU, S.S. ARAMĂ, ANCA STREINU CERCEL, RUXANDRA CĂLIN, C. BĂICUŞ, Impact of Highly Active Antiretroviral Therapy on Cytomegalovirus Viraemia in the Absence of Specific Anti-Cytomegalovirus Therapy ................................................. LUCIA AGOŞTON-COLDEA, TEODORA MOCAN, C. BOBAR, Arterial Stiffness and Left Ventricular Diastolic Function in the Patients with Hypertension ..................................................................................................................................... TEODORA MOCAN, LUCIA AGOŞTON-COLDEA, L.D. RUSU, RALUCA PAIS, M. GATFOSSE, L.C. MOCAN, M.L. RUSU, The Correlation Between Alcohol Consumption, Lipids, Apolipoproteins and Coronary Heart Disease ... BRÎNDUŞA DIACONU, TEODORA MOCAN, LIDIA CIOBANU, Risk Factors in Patients with Chronic Pancreatitis in Romania INIMIOARA MIHAELA COJOCARU, M. COJOCARU, R. TĂNĂSESCU, CECILIA BURCIN, ANDREEA CRISTINA MITU, IULIANA ILIESCU, LAURA DUMITRESCU, ISABELA PAVEL, ISABELA SILOSI, Detecting AntiProthrombin Antibodies in Young Women with Acute Ischemic Stroke ......................................................................... ALEXANDRINA L. DUMITRESCU, L. ZETU, SILVIA TESLARU, BEATRICE C. DOGARU, C.D. DOGARU, Is It An Association Betwen Body Appreciation, Self-Criticism, Oral Health Status and Oral Health-Related Behaviors? .........

305 313 323 331

337 343

CASE REPORTS CAMELIA IONESCU, MONICA ECOBICI, DANA OLARU, C. STĂNESCU, IOANA LUPESCU, M. VOICULESCU, Pneumoperitoneum – Rare Complication in End Stage Renal Disease Patient on Automated Peritoneal Dialysis .......... LAURA POANTĂ, D.L. DUMITRAŞCU, DANIELA FODOR, ADRIANA ALBU, Atrial Septal Aneurysm and Stroke – A Report of Two Cases .................................................................................................................................................... RALUCA TRIFĂNESCU, CĂTĂLINA POIANĂ, D. HORTOPAN, Autoimmune Thyroid Disease – A Continuous Spectrum GH. BURNEI, ANCA BURNEI, D. HODOROGEA, ŞT. GAVRILIU, ILEANA GEORGESCU, C. VLAD, LUCIA HURMUZ, DANA DAN, Renoureteral Diseases Inducing Hypertension in Children ....................................................................... SABINA ZURAC, R. ANDREI, G. PETSAKOS, LUCIANA NICHITA, ALEXANDRA BASTIAN, GIANINA MICU, ELIZA GRAMADĂ, CRISTIANA POPP, FLORICA STĂNICEANU, ŞTEFANA PETRESCU, GABRIELA NEGROIU, DORINA GIURCĂNEANU, VIRGINIA CHIŢU, Cutaneous Metastases of Malignant Melanoma – How Difficult Can It Be? ..........................................................................................................................................................

ROM. J. INTERN. MED., 2008, 46, 4, 267–378

351 357 361 367

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REVIEWS

Chronotherapy of Hypertension: When Can Be As Important As With What H. BĂLAN “Carol Davila” University of Medicine and Pharmacy IInd Medical Clinic, Clinical Emergency Hospital Ilfov County, Bucharest, Romania

The “manometric” way of considering the general management of high blood pressure (HBP) must remain ancient history. The huge therapeutical armamentarium (over 75 preparations, in 9 different classes) existing nowadays allows us to select the drugs most appropriate for the comorbidities of each case. The BP level target, unanimously considered a very important element of HBP management, must not be the only one. An important and very promising possibility to improve the therapeutical efficacy of the antihypertensive treatment is chronotherapy. By using ambulatory blood pressure monitoring (ABPM) it is now possible to “refine”, to better “tailor” the management of hypertension. Key words: chronotherapy, hypertension, circadian rhythms.

We are living in a universe in which rhythmicity is one of the main characteristics. The temporal variations of the cardiovascular functions have been remarked since the IInd century after Christ by Galen (quoted by 1). Many studies, in the past thirty years, have confirmed their existence, revealing that the maximal BP values are encountered in the morning (between 6.00 and 10.00 a.m.), a second peak being noted between 4.00 and 7.00 p.m.); between 03.00–04.00 a.m. are encountered the minimal values. It is now generally accepted that blood pressure (BP) represents a phenomenon with a temporal rhythmical variability. The variability of BP during a single day is huge: over 100.000 fluctuations [1]. The diagnostic and the therapeutical decisions in hypertension are still, to a great extent, based on the so-called casual/office/clinical BP values: the value at a certain casual moment, usually in the morning, after 10.00 o’clock, determined in medical settings. Maybe the most appropriate comparison of the reality and the reliability of a conclusion based on a single measurement at a single moment that can be completely undefinitory for the subject condition, is with a movie chronicle, based on a single frame. The human body is not only organized in space, anatomically, but it is also organized in time, in terms of biological rhythms. Circadian rhythms, and their alteration could be important ROM. J. INTERN. MED., 2008, 46, 4, 269–274

determinants of morbidity and mortality. They may also be very important in determining the responses to antihypertensive medications [2]. The primary goal of treatment of a hypertensive patient is to achieve the maximum reduction in long-term total risk of cardiovascular morbidity and mortality [3–6]. But the casual (clinical) BP values have many limitations: systematical overestimates (in at least 1/3 of the patients) = “white coat hypertension” (WCH); lack of precision in estimating the hypotensive effects of therapy; all the readings are made in artificial conditions: physical/psychic rest; offers no data during the sleep period. And also many sources of error: device-associated; observerassociated, even the presence of the observer = WCH; associated with the reading technique; “auscultatory gap” or the “pseudo-hypertension of the elderly” (Osler phenomenon). The better correlation of HBP prognosis with Ambulatory Blood Pressure Monitoring (ABPM) than with clinic/office/casual pressures can be attributed to a combination of two facts: first, the effect of the greater number of readings; the second factor is the more representative nature of the ambulatory readings. To counteract the effects of “the most active and efficient killer of our times” one must begin with the accuracy of the diagnosis, that can be achieved only by ABPM [7–14]. The most recent ABPM won the battles:

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♦ April 2003 – Medicare & Medicaid decided ABPM reimbursement for WCH. ♦ JNC VII (2003): “In most individuals, BP decreases by 10 to 20 percent during the night; those in whom such reductions are not present are at increased risk for cardiovascular events”. CHOBANIAN et al. [3]: “The normalisation of the circadian BP pattern to a dipper profile is a novel therapeutic goal, and accumulating medical evidence suggests this can delay the progression towards the renal and cardiovascular pathology known to be a consequence of the non-dipper BP pattern.” General situations where ABPM is extremely helpful: 1) hypertension in the elderly (WCH, systolic hypertension, pseudo-hypertension, hypotension, orthostatic/postprandial hypotension, diurnal/ nocturnal hypotension, drug induced hypotension); 2) autonomic dysfunction; 3) pulseless syndrome; 4) arrhythmias; 5) obese patients; 6) masked hypertension. N.B. Actigraphs or a diary concerning the type of activity, the sleeping/awakening hour, the moment of drug administration are mandatory as are also the AAMI (2002), BHS (1993), European Society of Hypertension Working Group on Blood Pressure Monitoring – International Protocol device validation. For all these reasons, ABPM is now considered the golden standard in: diagnostic, management, epidemiology, research, decisions concerning hypertension. The next generation of guides will be evidence based, not consensus based. Arguments in favour of ABPM: offers similar data to intra-arterial readings (the “golden standard”); has a good reproducibility; offers a clearly superior correlation with the cardio-vascular morbidity/mortality and with the echocardiographic, eye fundic examination, renal function evaluation; in populational studies it offers much more precise pressure/risk curves; highlights the real importance of the minor increases of BP; allows to avoid different kind of “artifacts”: WCH; “pseudohypertension” of the elderly (OSLER effect); placebo effect; masked hypertension; can have a good cost/efficiency ratio if we consider the economy generated by avoiding unnecessary therapy + his side-effects, allows to highlight the evolution of BP values in critical moments: the morning surge; the BP values during sleep. There are some special situations that have imposed ABPM: 1. to determine episodic, borderline

2

or uncontrolled HBP and orthostatic hypotension; 2. to establish the severity of HBP, in relation with TOD; 3. to determine the efficacy of the prescribed medication, regarding the doses and the frequency of their administration; 4. to establish the effects of the interaction of several drugs; 5. to certify the diagnosis of HBP. The timing of circadian (during 24 hours) BP peaks and troughs is quite predictable from one day to another in most people who adhere to a fairly regular sleep-activity schedule. Clearly, these data are compelling that human biochemistry and physiology are not constant; they rather vary in a predictable manner during the 24-hour time period. It seems plausible that timing of certain medical conditions and life-threatening emergencies may parallel these physiochemical circadian variations. There is an “evil crossing” of many unfavorable events early in the morning: Hemodynamic factors: BP and heart rate, stroke volume and cardiac output, total peripheral resistance, vascular tone, plasma volume, pulmonary arterial pressure, platelet agregability, blood coagulability, blood viscosity, serum cholesterol are increased; coronary blood flow, forearm blood flow, renal flow, glomerular filtration rate, urinary flow, fibrinolysis are decreased. Neuro-hormonal factors: autonomous tone, plasmatic concentrations of: norepinephrine, epinephrine, tyrosine, dopamine, prorenine, renine, angiotensin I, aldosterone, ACTH, cortisol, endogenous opioids have increased values; serotonine, natriuretic peptide system, Na excretion, K excretion have decreased values. In the first morning hours the physical and mental activity is increased, so that the “shear stress” of vascular atheroma is also increased, of sufficient magnitude to cause initial disruption of a stable atherosclerotic plaque. The term “dippers”describes those patients whose nocturnal pressure is at least 10% to 20% lower than their daytime pressure. “Extreme dippers” have a nocturnal pressure that is > 20% lower than their daytime pressure. In 10% to 30% of hypertensive patients, however, this characteristic pattern may be absent or blunted. These patients are known as “nondippers”. This subgroup of hypertensives are at the greatest risk for TOD (e.g., LVH, cerebrovascular disease, microalbuminuria, congestive heart failure, vascular dementia, myocardial infarction, renal damage, glucose

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intolerance, plasmatic fibrinogen increase, “procoagulant” trend in the morning). For the extreme dippers, the risk for TOD is also high (although not as high as with nondippers) [5][7–16]. The chronotherapy of hypertension takes into account the epidemiology of the BP pattern, plus potential administration-time determinants of the pharmacokinetics and dynamics of antihypertensive medications, as a means of enhancing beneficial outcomes and/or attenuating or averting adverse effects. Chronotherapy provides a means of individualizing treatment of hypertension according to the circadian BP profile of each patient, and constitutes a new option to optimize BP control and reduce risk. The development of new formulations of antihypertensive drugs, from short-acting to longacting to once-daily products, has raised the possibility of controlling the release of these drugs and timing it for when it is most effective. Many existing antihypertensive drugs accomplish this goal for hypertensive “dippers”. Treatment should also be directed toward converting hypertensive non-dippers into normotensive dippers. Some of the issues could be best addressed by administering antihypertensive drugs based on the principle of releasing their active agents at different times during the day, according to biological needs, which is the basis for chronotherapy. The ideal agent should produce a fall in BP only during the period that it is abnormally elevated (sleep in “nondippers”) and have little or no effect during the daytime when it is normal or close to normal. Evidence for the cardiovascular benefits of antihypertensive treatment is among the strongest in medicine. Even small reductions in BP for short periods substantially improve cardiovascular outcomes. More recent evidence has emphasized the importance of optimal blood pressure control, particularly on patients with high cardiovascular risk; the results are still unsatisfactory: 62% of cerebrovascular and 49% of ischemic heart disease can be attributed to suboptimal BP treatment. A 2002 poll of 200 primary care physicians treating hypertensive patients found that while 90% of them knew that circadian factors could boost patients’ risk of stroke or heart attack at certain times of the day only slightly more than half considered the body’s natural rhythms when prescribing treatment for hypertension. And while 80% knew of the increased early-morning risk of heart attack and stroke, only two-thirds told their

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patients to take their medication in the morning or when they woke up [18]. Twenty-four hour blood pressure control may be necessary to gain complete benefit from blood pressure-lowering therapy. Nighttime blood pressure is in general a better predictor of outcome than daytime pressure in hypertensive patients and the night-day blood pressure ratio predicts mortality, even after adjustment for 24-hour blood pressure [5–18]. An elevated BP during the sleeping hours causes LVH (gene expression in the heart is dramatically different during the day as compared to night), whereas a normal blood pressure during the sleeping hours allows LVH reduction. LVH is a cardinal manifestation of TOD in patients with hypertension and has a strong independent adverse prognostic significance. Reversing LVH is an extremely important goal of antihypertensive therapy. That means the risk/benefit ratio of a therapeutic intervention is not uniform across the 24 hour cycle but occurs in a diurnal fashion; the functional organization of the cardiovascular system shows specific time courses: a temporal organization is present in the clinical manifestations of most cardiovascular diseases; the sensitivity of the cardiovascular system to both pathological mechanisms and therapeutic interventions is timedependent. Normalization of the circadian BP pattern is considered to be an important clinical goal of HBP therapy because it may slow the advance of renal injury and avert end-stage renal failure. To alleviate/to abolish the morning surge is mandatory: the “evil crossing” during the morning hours generates a clustering of: silent myocardial ischemia, angina unstable pectoris, acute myocardial infarction (AMI), coronary spasm, pulmonary thromboembolism, ectopic beats, ventricular tachycardia, atrial flutter and fibrillation, paroxysmal supraventricular tachycardia, aortic aneurism rupture, spontaneous aortic dissection,strokes. All of them are much more frequent in the morning hours. It’s now possible to use either timecoordinated schedules of conventional antihypertensive drugs administration (see below) or products specially conceived for chronotherapy: Conventional antihypertensive drugs [3][10][17–21]: ♦ Any short acting BETA-BLOCKER should be administered in the morning; ♦ CALCIUM ANTAGONISTS WITH SUSTAINED RELEASE have a significant residual

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effect even after 24 hours: AMLODIPINE, ISOPTIN-RR, CALAN-SR, VERELAN-PM, ISRADIPINE, VERAPAMIL-SR. When you administer them in the morning you can attenuate the morning peak, but, by vesperal administration it is possible to induce a “dipper” profile, also attenuating the morning peak; ♦ Long/intermediate acting ACEI: it is preferable to administer them in the evening: Ramipril (in the Heart Outcome Prevention Evaluation (HOPE) trial the vesperal administration was found to be significantly associated with decreased cardiovascular morbidity/mortality in patients at high risk for cardiovascular events: combined cardiovascular endpoint (cardiovascular mortality, MI, stroke) – a decrease of 22% (p < 0.001); cardiovascular mortality – a decrease of 26% (p < 0.001); stroke – a decrease of 32% (p < 0.001). It is preferable to use morning administration for: ♦ Quinapril (it is possible to have an excessive effect with a vesperal administration) ♦ Perindopril (for an effect longer than 24 hours) ♦ DIURETICS: Indapamide; Xipamide have both similar BP lowering effects in morning or evening administration ♦ α-BLOCKERS: DOXAZOSIN – determine the higest BP reduction when is administered in the morning. Dippers, non-dippers and excessive dippers seem to get some benefit from chronotherapeutic dosing.” [16]. But both silent cerebrovascular damage and anterior ischemic optic neuropathy are potential risks of excessively low overnight blood pressure. Also, extra-low night time blood pressure dips mean an even steeper early morning blood pressure surge. For these patients who have “excessive dipping” at night, their risk of excessive dropping blood pressure overnight could increase. Chronotherapeutic antihypertensive drugs, which are designed to have their peak effect after the overnight risk period, may be an optimal therapeutic option in vesperal administration. HBP CHRONOTHERAPY MUST BE MODULATED AND VERIFIED BY ABPM

Drug toxicity, drug activity and drug kinetics, chronotoxicology, chronopharmaco-dynamics and chronokinetics have all biological rhythms. All

4

mentioned in the next paragraph have biological rhythms: the gastro-intestinal tract, its pH, the digestive motility, activity/rest, posture, blood flow, tissular perfusion, the activity of the hepatic enzymes, the hepatic blood flow, the glomerular filtration rate, urinary pH, the absorption (oral, parenteral, cuteneous, transmucosal), distribution (plasma protein binding, tissular binding, volume of distribution), metabolism of drugs (in the liver, in other tissues), elimination of drugs (renal, biliary, other ways). There are many sources of variability in chronopharmacokinetics: pharmacological variability depends on: the patient (elderly, children, male/female); the pathology (renal failure, hepatic failure, cardiac failure, immuno-depression, malnutrition, obesity); the drug (route of administration, condition of administration: single dose, repeated doses, constant rate infusion). Despite this huge complexity of internal and external influences, chronotherapy has already won many battles: morning administration of corticosteroids; asymmetrical administration of corticosteroids in Addison disease; vesperal administration of H2 receptors blockers; vesperal administration of HMG-CoA antagonists; vesperal administration of Theophylline retard preparations; asymmetrical administration of beta-2 antagonists in asthma; temporal administration of melatonin in sleep disturbances treatment; vesperal administration of aspirin in preeclampsia. Chronotherapy is a must: when the pathologyrisk is well-known; when the effect of the drug can be obtained only by a time-modulated therapy; when the therapeutic “window” is small; when the kinetics and/or the drug effects are time-dependent; when the treatment must mimic the temporal structure of a function. Main targets of hypertension treatments [1][3][9]: to alleviate/abolish the morning peak; to induce a benefic circadian profile (“dipper”); to increase the action period (“missing pill”); to avoid significant hypotension; effective 24-h BP control; smooth antihypertensive effect with reduced variability; attenuation of the early morning surge in BP; maintenance of the normal circadian pattern of BP; effective therapeutic coverage in the face of suboptimal compliance;lack of reflex activation of the sympathetic nervous system; to have the lesser and the most discrete possible side effects; lack of unfavorable metabolic effects; to be efficient in monotherapy, in single administration; to decrease the local and general vascular tonus; to maintain

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Chronotherapy of hypertension

unchanged the cardiac output and the local blood flow; to allow the synergic association, without side effects, with other antihypertensive drugs. Questions to be asked: can the BP rhythmicity be modified in such a way to modify the morbidity/ mortality?; is it possible to personalize chronotherapy?; is it worth individualizing the chronotherapy of hypertension?; when is it worth doing it? To produce its characteristic effects a drug must be present in appropriate concentrations at its sites of action at the right moment [Cmax, Tmax, Vd, AUC, bioavailability, binding to plasmatic proteins, T1/2 – all have time-dependent variability [9]]. New, innovative drug-release technologies, allowing medications that are maximally effective at different points in our 24-hour circadian cycle to be used according to the body's specific physiologic needs have been developped. This new era of therapeutics has been termed chronopharmacology or chronotherapy. Drugs specially conceived for CHRONOTHERAPY: an efficient 24 hours control, the highest reductions appearing in the critical hours of the morning; their action begins after 4 hours; their maximal plasmatic levels are reached around 10.00 a.m. N.B. These kind of preparations must be swallowed whole and not chewed, crushed, or split, as this may damage the drug delivery system and cause dose dumping. Chronotherapy of hypertension: special formulations marketed in the USA: COVERA-

HS; VERELAN-PM; CARDIZEM-LA; INNOPRANXL; NIFEDIPINE-GITS; PROCARDIA-XL and conventional extended release medications dosed at bedtime versus upon awakening (FDA recommendations). “You may be getting the idea that all you need to do is give patients drugs once-a-day drugs at night rather than early in the morning on the grounds that they will have a nice drug level when they wake up in the morning for the dangerous morning hours, but that is not necessarily the case.While this is appropriate for drugs designed specifically for this purpose, just giving a conventional drug at night may bring blood pressure down too low at night and end up having little effect for the rest of the day. You have to know about the properties of the individual drug when giving it in the morning or in the evening, and just simply giving all drugs in the evening is not desirable. Attempts to oversimplify chronotherapy for hypertension could have dangerous results.” [1][4]. Chronotherapy is now possible and constitutes a new option to optimise BP control and reduce cardiovascular risks. Significant administration-time differences in the kinetics and in the beneficial and adverse effects of antihypertensive medications are well known. The first steps have been made. Let’s imagine the following ones. Don’t forget: “When there’s a will… there’s a way”.

Abordarea strict manometrică a hipertensiunii arteriale (HTA) trebuie să devină istorie. Impresionantul arsenal terapeutic actual (peste 75 produse, în 9 clase diferite) ne permite să alegem medicamentele cele mai adecvate comorbidităţii pacientului. Nivelul optim al TA, unanim considerat un element foarte important al abordării HTA nu trebuie să fie singurul scop. O importantă şi foarte promiţătoare posibilitate de a ameliora eficacitatea terapeutică a medicamentelor este cronoterapia. Prin utilizarea monitorizării ambulatorii automate este acum posibil “a rafina”, “a croi” mai bine abordarea terapeutică. Corresponding author: H. Bălan, Assoc. Professor Emergency Hospital Ilfov County 49–51, Blvd.Basarabiei, Bucharest, Romania E-mail: [email protected].com

REFERENCES 1. 2.

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BĂLAN H., Hipertensiunea arterială din perspectiva cronobiologică. Ed. InfoMedica, 2002. LEMMER B., The chronopharmacology of cardiovascular medications. Ann. Rev. Chronopharmacol., 1984, 2, 199–258.

274 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

H. Bălan

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CHOBANIAN et al., The seventh Report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure. JAMA, 2003: 289:2560–2571. PICKERING T., ALPERT B.S., DE SWIET M. et al., Ambulatory blood pressure. Biophysical measurement series, 1994. COHUET G., STRUIJEKER-BOUDIER H., Mechanisms of target organ damage caused by hypertension; therapeutic potential. Pharmacol. Ther., 2006, 111 (1): 81–98. BAKRIS G., HILL M., MANCIA G. et al., Achieving blood pressure goals globally: five core actions for health-care professionals. A worldwide call to action. J. Hum. Hypertens., 2008, 22 (1): 63–70. NADAR S.K., TAYEBJEE M.H., MESSERLI F., LIP G.Y., Target organ damage in hypertension: pathophysiology and implications for drug therapy. Curr. Pharm. Des., 2006; 12 (13): 1581–92. BOGGIA J., LI Y., THIJS L. et al., The International Database on Ambulatory blood pressure monitoring in relation to Cardiovascular Outcomes (IDACO) investigators. Prognostic accuracy of day versus night ambulatory blood pressure: a cohort study. Lancet, 2007; 370:1219–1229. PORTALUPPI F., SMOLENSKY M.H. (eds), Time-dependent structure and control of arterial blood pressure. A Symposium of the New York Academy of Sciences. – N.Y.: New York Academy of Sciences, 1996. PARATI G., STAESSEN J.A., Day-night blood pressure variations: mechanisms, reproducibility and clinical relevance. J. Hypertens., 2007, 25 (12): 2377–2380. PICKERING T.G., The clinical significance of diurnal blood pressure variations: Dippers and non-dippers. Circulation, 1990, 81:700–702. VERDECCHIA P., ANGELI F., GATTOBIGIO R., RAPICETTA C., REBOLDI G., Impact of blood pressure variability on cardiac and cerebrovascular complications in hypertension. Am. J. Hypertens., 2007, Feb.; 20 (2): 154–61. VERDECCHIA P., ANGELI F., SARDONE M. et al., Is the definition of daytime and nighttime blood pressure prognostically relevant? Blood Press Monit., 2008, 13 (3): 153–155. BEN-DOV I.Z., KARK J.D., BEN-ISHAY D., Predictors of all-cause mortality in clinical ambulatory monitoring: unique aspects of blood pressure during sleep. Hypertension, 2007, 49 (6) 1235–41. FAGARD R.H., CELIS H., THIJS L. et al., Daytime and nighttime blood pressure as predictors of death and cause-specific cardiovascular events in hypertension. Hypertension, 2008, 51 (1): 55–61. VERDECCHIA P., Prognostic value of ambulatory blood pressure: current evidence and clinical implications. Hypertension, 2000; 35: 844–851. HERMIDA R.C., AYALA D.E., PORTALUPPI F., Circadian variation of blood pressure: the basis for the chronotherapy of hypertension. Adv. Drug Deliv. Rev., 2007; 59 (9–10): 904–22. MANCIA G., Blood pressure reduction and cardiovascular outcomes: past, present, and future. Am. J. Cardiol., 2007, 6; 100 (3A): 33–91. HERMIDA R.C., AYALA D.E., CALVO C., PORTALUPPI F., SMOLENSKY M.H., Adv. Drug Deliv. Rev., 2007, 31; 59 (9–10): 923–39. TOUITOU Y., HAUS E. (eds), Biological rhythms in clinical and laboratory medicine. Berlin, Springer-Verlag, 1992. REDFERN P.H., LEMMER B. (eds), Physiology and pharmacology of biological rhythms. Berlin, Springer-Verlag, 1997; 375–414.

Received July 12, 2008

Cardiovascular Risk in Patients with Peripheral Vascular Diseases ADINA STOICA, CARMEN GINGHINĂ “Prof. Dr. C.C. Iliescu” Institute for Cardiovascular Disorders, Bucharest, Romania

Peripheral arterial disease (PAD) is characterized by increased incidence and prevalence, both of which increasing with age (prevalence about 19% above 70 years versus 2.5% below 60 years); PAD is strongly dependent upon smoking and diabetes mellitus as cardiovascular risk factors. Patients suffering from peripheral arterial disease are patients at high cardiovascular risk, albeit symptomatic patients (intermittent claudication) or asymptomatic patients (added risk for cardiovascular events 4–5%/year); these risks increase in patients needing revascularization surgery (6%), mainly due to association of coronary or cerebro-vascular disease. A fair assessment of preoperative risks (risks depending on patient background, type of surgery, emergency status of surgical procedure) aids in the optimal management of such patient both at the time of performing the procedure and in the long-term patient management. This is why new parameters are required for assessing the risk and identifying a subgroup of patients at high-risk for acute cardiovascular events and for customizing the diagnostic and therapeutic algorithms for such patients.

It has been previously proven that peripheral vascular disease is an independent predictor for cardiovascular death, more important in assessing survival rates than the previous clinical history of coronary heart disease [1]. Patients with peripheral arterial disease are at high risk for cardiovascular morbidity and mortality, regardless of sex, albeit symptomatic patients (intermittent claudication) and asymptomatic patients (Fig. 1). Leng et al. showed in a prospective study including 1592 patients with claudication and asymptomatic aged 55–74 years, followed for 5 years that asymptomatic patients seem to have increased risk rates for cardiovascular events and death similar to the ones of symptomatic patients [2]. Even following successful myocardial revascularization, patients also suffering from peripheral arterial disorders remain at high mortality rates (OR–2.77) as compared to the patients with myocardial revascularization therapy but with no peripheral arterial disorder [3]. Since the characteristic symptom – and namely intermittent claudication – is present in at most 30% of patients (40–50% are either asymptomatic or present with atypical complaints – paresthesia, tingling, numbness in the affected limb) [4], and the effort capacity is most frequently decreased, the diagnosis of limb arteritis is most often either omitted or late, such patients showing increased risk for cardiovascular events and lacking secondary prevention measures. ROM. J. INTERN. MED., 2008, 46, 4, 275–283

The early diagnostic of peripheral vascular disease and the optimal management of such patients may decrease the cardiovascular morbidity risk since although peripheral revascularization (surgical/interventional) may improve symptoms, they show no impact on survival rates [4]. INCIDENCE AND PREVALENCE OF PERIPHERAL ARTERIAL DISEASE

The incidence and prevalence of peripheral arterial disease depend on age, risk factors in study group, association with other atherosclerotic disorders, the diagnostic methods for peripheral arterial disease (questionnaire, clinical examination, ankle-brachial pressure index, continuous Doppler examination), so that they may vary among different studies (Table I) [1][2][5–10]. Peripheral arterial disease is more frequent in elders (prevalence about 19% in age group over 70 years versus 2.5% below 60 years) and is more frequent in men. Epidemiology and clinical consequences of the disease are closely related to the atherosclerotic risk factors (smoking, diabetes, hypertension, dislipidemia, family history, hyperhomocysteinemia, post-menopause status), since the most frequent underlying cause of the disease is atherosclerosis. Amongst all risk factors, smoking is a strong predictor of peripheral arterial disease, the risk for developing

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Adina Stoica, Carmen Ginghină

2

Normal subjects Asymptomatic patients Symptomatic patients

Patients with severe symptoms

Fig. 1. – Peripheral arterial disease patient’s mortality by symptoms (adapted by Criqui M.H. et al., N. Engl. J. Med., 1992; 326:381–386). Table I Epidemiologic characteristics of peripheral arterial disease patients Trials

Characteristics

Diagnosis

Framingham

2336 men + 2873 women

Rose questionnaire + clinical exam at 2 years

Age (years) 30–44

65–74

Edinburgh

1592 people

Questionnaire + ABI

55–74

Incidence

Prevalence

Notes

6/10000 men /year 3/10000 women / year 61/10000 men/year 54/10000 women/year 15.5/1000/ year

4.5%

Prevalence of CHD2 – 11.1%

1

Criqui et al. (San Diego Study)

613 people

Questionnaire + clinical exam + ABI + continuous Doppler ultrasound

PARTNERS

6979 people

ABI

NHANES

2174 people

ABI

< 60

2.5%

60–69

8.3%

> 70 > 70 or 50– 69 for smokers/ diabetes mellitus > 40 > 70

18.8% 29%

4.3% 14.4%

16% has ATS3 in other vascular territory

3

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Table I (continued) Rotterdam

7715 people

Rose Questionnaire + ABI

Hooi et al.

2327 people

Rose Questionnaire + ABI

Dawson et al.

Leng et al.

1592 people

Bowlin et al.

10059 people

> 55

9.9/1000/year (1/1000/year had symptoms) 40–59 60–69 > 70 55–74

Rose Questionnaire + ABI

19.1% (6.3% had symptoms)

40–65

3% 8% 19% 1.6% /year with symptoms 8.6 % /year with symptoms

ABI – ankle-brahial index, CHD – coronary heart disease, ATS – atherosclerosis

peripheral arterial disease being 2–3 times higher in smokers than the risk for developing coronary heart disease [6], while diabetes mellitus increases the risk for critical ischemic complications and amputations (RR=7–15 according to various authors) [1][8], so that the risk factor profiles in study subgroups influence both epidemiology data (incidence, prevalence), and the morbidity and mortality rates. ASSOCIATION WITH CORONARY AND CEREBROVASCULAR DISEASES

The prognosis for patients with peripheral vascular disease is altered by increased risk for ischemic cardiovascular events due to simultaneous coronary and cerebrovascular diseases, most frequently due to atherosclerosis [11]. The recorded prevalence of coronary and cerebrovascular disease in patients suffering from obliterating arteritis depends on the diagnostic criteria and methods used. About one third up to one half of the patients with obliterating arteritis present obvious signs of coronary heart disease in physical examination and resting ECG and up to two thirds of these patients present alterations of stress tests [12]. A large proportion (60–80%) of patients with peripheral arterial disease present significant coronary lesions as proven by angiography, in at least one vascular territory [13]. These patients may also associate carotid artery lesions which may be significant in 12 up to 25% according to various authors [14]. Various trials identified simultaneous alterations in several vascular territories in large proportions. The CAPRIE study (about 19000 patients in the study group) identified that 1 of

4 patients with atherosclerotic lesions presented lesions in several vascular territories [15]. Also, the AGATHA study including 8891 subjects identified a secondary affected vascular territory in patients with peripheral arterial disease in about 13% of patients (simultaneous peripheral arterial disease and coronary heart disease – 6.7% and simultaneous peripheral arterial disease and carotid artery lesions – 6.2%), while simultaneous alterations in all three territories were identified in 7.1% of all subjects [16]. A high rate of association with peripheral arterial disease was also noted in the groups with coronary heart disease and cerebrovascular disease (1/3 of men and 1/4 of women with known coronary heart disease or cerebrovascular disease also show peripheral vascular lesions) [17]. The REACH registry prevalence rate for peripheral vascular disease in coronary heart disease patients was 11% [18]. CARDIOVASCULAR EVENTS RISK

More recent trials [19–21] reported combined risks for AMI, stroke and death reaching 4–5% /year, with no significant differences due to sex or intermittent claudication as compared to asymptomatic patients, but increasing up to 6% /year when also including patients with peripheral revascularization therapy. The cardiovascular risk is increased in patients with severe symptoms of ischemia (critical ischemia – 1-year mortality rate – 25%) and with a decreased ankle-brachial pressure index [22]. Several authors proved that the relative risk for cardiovascular mortality and morbidity (coronary heart disease, stroke) is higher in patients with peripheral vascular disease than in patients with no

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arterial disease, regardless of the clinical evidence pleading for coronary heart disease. Thus Eagle et al. proved on a group of 2296 patients with peripheral arterial disease vs. 13953 with no arterial disease, all of them suffering from stable angina pectoris that the mortality rate was 25% higher in patients with peripheral vascular disease [23], while the incidence of stroke was 42% higher in patients with peripheral arterial disease [24]. A high risk for cardiovascular events and mortality was also identified in patients without evidence of coronary heart disease; Yogt et al. showed not only a high risk for coronary heart disease (RR–3.7) and for cardiovascular events (RR–4), but also increased risks for overall mortality (RR–3.1 in 4 years). Other authors also documented an increased risk for cardiovascular and coronary heart disease mortality, Criqui et al. identifying a relative risk for cardiovascular mortality 5.9 times higher and a relative risk for coronary heart disease mortality 6.6 times higher in these patients; moreover, they also proved that the cardiovascular mortality risk increases furthermore in patients with severe symptoms due to lesions in large vessels – distal aorta, iliac arteries (RR–15)[25]. Other authors also correlated the severity of peripheral arterial disease with increased risk for cardiovascular events (AMI, stroke) and cardiovascular death [1]. Peripheral revascularization surgery therapies include high risks for post-surgical cardiovascular events and mortality. Mortality is 25–50% higher in patients with cardiac disorders subject to high-risk surgical procedures than in normal-hearted patients. Postoperative risks partly depend on the type of surgical intervention. The elective aortobifemoral revascularization interventions associate an in-surgery mortality rate of 3.3% and a cardiovascular morbidity rate of 8.3%, with acute myocardial infarction (incidence between 0.8 and 5.2% in various studies) and kidney failure (0–4.6%) as the leading cardiovascular complications [26]. For interventions below the inguinal region in selected patients, mortality reached up to 6% in various studies, risk significantly increasing for amputations. Major lower limb amputations associated a high risk for cardiovascular mortality in 30 days of up to 30% and morbidity rates (AMI, stroke and infections) ranging from 20 to 37% according to various authors [27]. Revascularization of the aorta and below the inguinal fossa is still associated with increased 30-day and 1-year mortality rates according to Fleischer. He

4

moreover proves that aortic surgery associates increased short-term mortality, while interventions below inguinal fossa are most often associated with increased long-term mortality [28]. Krupski et al. also proved increased 2-year risk for fatal / nonfatal myocardial infarction in patients with infrainguinal by-pass versus aorto-bifemoral by-pass (RR–3.5), differences being due to increased prevalence of diabetes, coronary heart disease (previous old myocardial infarction, angina pectoris) and heart failure in patients with infrainguinal peripheral vascular disease [29][30]. ESTIMATING CARDIOVASCULAR RISK

Perioperative cardiovascular risk depends on the patient and on the type of surgical procedure, as well as on the circumstances leading to surgical procedure (emergency / elective surgery). During the surgical intervention the patient suffers disturbances of the cardiovascular system mainly due to the anesthesia and to the surgical procedure (depressed myocardial contractility, increased myocardial excitability, indirectly due to respiratory distress, blood pressure variations, variations of the ventricular filling pressure, of the blood volume and temperature, effects of tonus variations of the vegetative nervous system) or due to the complications which additionally over-stress the cardiovascular system (blood loss, cardiac complications – AMI, arrhythmias, acute pulmonary edema, heart failure, pulmonary embolism, pulmonary/systemic infections, respiratory/kidney complications, etc.). The perioperative risk is therefore partly influenced by factors pertaining to the type of intervention (hemodynamic changes induced by the aortic clamp, water and electrolyte balance alterations, surgical technique), the duration of the surgical procedure (complication risk following the correction according to the type of intervention does not seem to correlate with the duration unless major complications occur), the emergency status of the procedure (AMI/death risk increases 2.5– 4 times as compared to elective surgery). The factors pertaining to the previous disease history significantly influence the perioperative prognosis, since revascularization surgery highrisks are mainly due to the association with coronary heart disease, justifying the interest in a thorough assessment of all patients before surgery in order to identify all potentially-correctable risk

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factors in order to improve the short-term and longterm prognosis. There has been previous concern for identifying some independent pre-operative predictors significantly correlated with increased risk for fatal or potentially fatal peri-operative complications noted in non-cardiac surgical procedures on patients aged over 40 years. Various authors identified several clinical factors (based from personal history, physical examination and resting ECG) predictive for perioperative risk factors (coronary heart disease – AMI in previous 6 months or unstable angina in the previous 6 months, angina (Canadian class III or IV), congestive heart failure – protodiastolic gallop, turgescent jugular veins or history of acute pulmonary edema, non-sinus rhythm, more than 5 premature ventricular complexes per minute, severe aortic stenosis, creatinine levels exceeding 2mg/dl, chronic liver disease or diabetes, age over 70 years, the type of surgical intervention – aortic surgery, the emergency status)[31–33]. Based on these factors a multifactorial index of cardiac risk in non-cardiac surgical procedures was drafted – the original index – Goldman [31]. Since the index was derived based on unselected patient studies, aged over 40 years, the index seems to underestimate by almost 40% the risk in patients at high basal risk (i.e. patients with stable angina, in which the risk is 2–4% even if lacking any other risk factor) or in patients undergoing more complex surgical procedures (i.e. aortic surgery)[34]. More recent works suggested criteria for assessing the surgical risk simpler than the composite risk score, factors which proved by multivariate analysis to lead to increased perisurgical risk for myocardial infarction, heart failure or death; they were classified as major, intermediate and minor risk predictors [35]. The major risk predictors are: − Coronary syndromes: − acute or recent MI (more than 7 days, but less than 1 month) with evidence of increased ischemic risk either clinically or at nonivasive procedures − unstable or severe angina (Canadian class III–IV) − Decompensated heart failure − Severe arrhythmias: − high-degree atrio-ventricular blocks − symptomatic ventricular arrhythmias in the presence of heart disease − supraventricular arrhythmias with uncontrolled heart rates



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Severe valvular disorders (severe aortic stenosis) Intermediate risk predictors: − moderate angina pectoris (Canadian class I–II) − previous myocardial infarctions (history or pathologic Q waves) − compensated or previous heart failure − diabetes mellitus (especially if insulin-dependent) − kidney failure (creatinine levels above 2 mg/dl) Minor risk predictors: − old age (more than 70 years) − ECG modifications (left ventricular hypertrophy, left anterior fascicular block, ST-T changes) − Non-sinus rhythm (i.e. atrial fibrillation) − decreased exercise capacity − previous history of stroke − uncontrolled hypertension Depending on the presence of risk factors the attitude is expected to be adjusted; therefore, for major risk predictors one may opt for canceling or at least delaying the surgical procedure until the patient is stabilized, if the respective procedure is not an emergency, while if only intermediate risk predictors are present (markers associated with increased risk for peri-surgical heart complications one may perform a more thorough preoperative assessment using noninvasive techniques and, depending on the results, using invasive explorations (coronary angiography). In the presence of minor risk predictors the patients are able to undergo the surgical procedure or, in case of decreased tolerance to effort (less than 4 METs), they should be non-invasively assessed and, depending on the results of the later investigations, they may also undergo coronarography (Fig. 2) [35]. Therefore, following clinical assessment, the patients may a) avoid surgical procedure (major risk predictors), or b) undergo surgical procedures (minor risk predictors) or c) require noninvasive and coronary angiography before undergoing the surgical procedure depending on the increased risk factors involved, explorations which may lead to performing coronary revascularization or intensive medical therapy prior to elective peripheral revascularization. It was moreover proved that the clinical factors are more sensitive than the surgical factors in predicting peri-surgical complications. Several long-term post-surgical revascularization studies (2 to 5 years follow-up) have been performed on patients with peripheral vascular disease. Such studies identified that the longterm risk assessment for patients undergoing surgical revascularization may consider all clinical risk

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6

Fig. 2. – Stepwise approach to preoperative cardiac assessment at peripheral arterial disease patients. (adapted by Eagle K.A. et al., ACC/AHA Guideline Update on Perioperative Cardiovascular Evaluation for Noncardiac Surgery, Circulation, 2002; 105:1257).

markers: ejection fraction, dobutamine stress echocardiography, stress scintiscan with thaliumdipiridamol, coronarography. Long-term heart disease mortality for patients undergoing surgery increases in the presence of major clinical risk factors, of decreased ejection fraction (EF lower than 35%), of multiple ischemic segments in stress echocardiography

or stress scintiscan, or of multi-vascular coronary heart disease. The best cost-efficiency ratio considering the 5-year event-free interval was deemed to be the stress scintiscan with thalium-dipiridamole for intermediate clinical risk patients; patients at high risk require coronarography assessment while

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Cardiovascular risk in peripheral vascular diseases

low-risk patients investigations [36].

do

not

require

further

FUTURE DIRECTIONS

The worldwide general interest noted in the past couple of years in prevention strategies is in identifying new cardiovascular disease/cardiovascular event risk stratification markers, in healthy population as well as in patients with proven cardiovascular disorders (hypertension, coronary heart disease, acute coronary syndromes), in order to identify population groups at risk, which may benefit from optimal treatment. The most promising results amongst the latest studied parameters promising results seem to arise from studying certain inflammation markers (i.e. C reactive protein, interleukins IL1, 6,18) and the endothelial dysfunction (intima-media index, flowmediated vasodilation). It is accepted that using such assessment methods still requires wider studies in order to pinpoint their role in the risk stratification strategies for patients with coronary heart disease and stable/unstable angina, while little evidence is available for patients with peripheral vascular disease [37]. The increased prevalence of peripheral vascular disease in older patients (8% between 60 and 69 years and 19% in patients aged over 70 years) and the increased risk for cardiovascular and coronary heart disease mortality in the latter patients tend to justify the interest in identifying new high-risk identification markers for cardiovascular events in such patients. Moreover, in

281

patients suffering from peripheral vascular disease, the methods available for diagnosing simultaneous or successive timely alterations of other vascular territories (i.e. coronary or cerebral arteries) are somewhat limited by patients symptoms (stress test) or by available resources (stress scintiscan, stress echocardiography) or even pose higher risks for complications in such patients – invasive maneuvers (coronarography). This is why the identification of new parameters is required for a simpler, more cost-effective assessment of cardiovascular event risk; such parameters are required in the peri-surgical and long-term risk assessment algorithm for such patients. An accurate assessment of the cardiovascular risk of peripheral vascular disease patients assists in lowering the cardiovascular event risk (a) on the short run via (a.1.) pre-surgical measures (optimization of medical treatment and possibly delaying the surgical intervention until compensating/correcting heart disorder and/or any other associated disorders or, in special circumstances – myocardial or carotid artery revascularization – albeit interventional or chirurgical) and (a.2.) intra-operative measures (i.e. careful intraoperative monitoring, including invasive monitors, the choice of an adequate surgical technique, lowering the blood loss and hemodynamic alterations due to aortic clamping), as well as (b) on the long run via secondary prevention measures. The optimal management of patients with peripheral vascular disorder may lead to lowering the cost of short-term and long-term care since the social costs are much higher for these patients.

Boala arterială periferică se caracterizează printr-o incidenţă şi prevalenţă care cresc cu vârsta (prevalenţa de aproximativ 19% la grupul de vârstă de peste 70 de ani comparativ cu 2,5% la grupul de vârstă sub 60 de ani) şi este puternic asociată cu fumatul şi diabetul zaharat dintre factorii de risc cardiovasculari. Pacienţii cu arteriopatie periferică reprezintă un grup de pacienţi cu risc cardiovascular crescut; atât pacienţii simptomatici prin claudicaţie intermitentă, cât şi cei asimptomatici (risc de evenimente cardiovasculare de 4–5%/an), risc care creşte la pacienţii care necesită revascularizare chirurgicală (6%), risc crescut din cauza asocierii bolii coronariene sau cerebrovasculare. Estimarea corectă a riscului preoperator (risc ce depinde de terenul pacientului, tipul intervenţiei chirurgicale şi caracterul de urgenţă al intervenţiei) ajută la un management optim al acestor pacienţi atât perioperator, cât şi pe termen lung. De aceea este necesară găsirea unor noi parametri de evaluare a riscului care să ajute la identificarea unui subgrup de pacienţi cu risc foarte înalt de a suferi un eveniment cardiovascular acut şi individualizarea algoritmului de diagnostic şi a tratamentului la aceşti pacienţi.

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Corresponding author: Adina Stoica “Prof. C.C. Iliescu” Institute for Cardiovascular Disorders 285 Şos. Fundeni E-mail: [email protected]

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7. 8. 9. 10. 11. 12.

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24. 25.

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Photodynamic Therapy – Indications and Limits in Malignant Tumors Treatment ADRIANA GABRIELA FILIP1, SIMONA CLICHICI1, DOINA DAICOVICIU1, DIANA OLTEANU1, ADRIANA MUREŞAN1, SIMINA DREVE2 1

Physiology Department, “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca 2 National R&D Institute of Isotopic and Molecular Technologies, Cluj-Napoca, Romania

Photodynamic therapy (PDT) is a very promising technique used for the treatment of a variety of solid neoplasms, based on the formation of singlet oxygen induced by a photosenstizer after irradiation with visible light. The mechanism of interaction of the photosensitizers and light is discussed, along with the effects produced in the target tissue. PDT has been approved in many countries for the treatment of lung, esophageal, bladder, skin and head and neck cancers. The antitumor effects of this treatment result from the combination of direct tumor cell photodamage, destruction of tumor vasculature and activation of an immune response. The present status of clinical PDT is discussed along with the newer photosensitizers being used and their clinical roles. Despite the promising results from earlier clinical trials of PDT considerable additional work is needed to bring this new modality of treatment into modern clinical practice. Key words: photodynamic therapy, photosensitizers, cancers, side effects.

Despite the latest advances in oncology, the problem of treating malignant diseases has not been resolved yet. In most cases, the treatment is beneficial at early stages of cancer. However, two thirds of patients reveal advanced cancer and only half of them undergo special treatment. Surgery, radiotherapy, and combined treatment have limited capabilities for advanced cancer, most of the patients die of relapses and metastases. Moreover, many patients (up to 25 percent) have operable cancer, but cannot have surgical treatment. This is because of serious associated diseases and agerelated disorders. These patients often undergo organ-saving surgical treatment with a high rate of local relapses. Until the last decade, there was no adequate treatment for these patients either. The advent of photodynamic therapy (PDT) has considerably extended oncologic capabilities. PDT is a relatively new therapeutic modality for neoplastic diseases, which involves light activation of certain photosensitizers (PS) that have been somewhat selectively taken up by the target tissue in the presence of molecular oxygen. PS are compounds that absorb energy from light of specific wavelengths and are capable of using that energy to induce reactions in other non-absorbing molecules [1]. PDT offers a number of advantages over traditional therapies for malignant tumors. First, PDT is highly selective and targeted in action. Second, it is free of surgical risks, serious damages, and systemic ROM. J. INTERN. MED., 2008, 46, 4, 285–293

complications. Third, PDT sessions can be repeated as many times as needed. Fourth, a single PDT procedure enables both the treatment and fluorescent diagnosis. Finally, most patients exhibit tumor resolution after a single PDT session, which can be performed under outpatient conditions [2]. HISTORY OF PDT

The studies regarding PDT have a long history, which dates back more than a century. The first clinical application of PDT was mentioned in 1903, when two researchers, Von Tappeiner H. and Jasionek A., observed that basal cell tumors would heal when exposed to eosine and light for a few weeks [3]. In 1942 Auler and Banzer injected hematoporphirin in tumor carrying animals and found that after exposing them to a halogen lamp, fluorescence and tumor necrosis appears. The purification of hematoporphirin, the use of its derivatives, and especially the introduction of laser as a light source by Maiman in 1960 lead to an improvement of the effects of this therapy [4]. The development of endoscopy offered new opportunities for the treatment of tumors located in less accessible areas such as the esophagus, the lung, the bladder, Hayata et al. being the first to utilize laser in the treatment of bronchial tumors [4]. In the USA Photofrin is the only PS approved by

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the Food and Drug Administration (FDA) for clinical use in palliation of “patients with completely obstructing esophageal cancer and for treatment of microinvasive endobronchial nonsmall cell lung cancer in patients for whom surgery and radiotherapy are not indicated” (National Cancer Institute http://cancernet.nci.nih.gov). Because of the long lasting skin phototoxicity of Photofrin, several new PS have recently been introduced in clinical trials: 5 amino-levulinic acid (5–ALA, Levulan; DUSA Pharmaceuticals Inc. Wilmington, MA), the methyl ester of ALA (Metvix, Photocure ASA, Oslo, Norway), Visudyne (verteporfin, benzoporphyrin derivative monoacid ring A, BPD-MA) and mesotetra-hydroxylphenyl-chlorin (mTHPC, Foscan, Biolitec Pharma Ltd., Dublin, Ireland) [2]. PRINCIPLE OF PDT

PDT involves the administration of a PS followed by local illumination of the tumor with light to activate the specific drug so that PS would pass from the base state to the excited state of singlet, with higher energy and very short life [2]. From this state the substance can follow two paths: it either passes back to the unexcited, base form and emits fluorescence which allows the detection of the photosensitiser in the tissue and the visualization of the tumor [5], or it passes in a triplet state with low energy and longer life span than the singlet form. In a triplet state, the molecule transfers its own energy either to the molecular oxygen present in the tissues and generates 1O2 (reaction type II) [4], or to the biomolecules from the tissue and it generates hydroxyl radicals, hydrogen peroxide, and anion superoxide (reaction type I) [5]. Radicals can also be formed after the reaction of 1O2 with the proteins and the unsaturated lipids from the cells resulting hydroperoxides, which in their turn react as secondary reactive oxygen species (ROS). The radicals and the peroxides having a long life can diffuse in the cell and can determine distant oxidative lesions, even in the DNA or proteins [6–8]. In fact, the results of the in vivo application of PDT depend on the effects that it has on tumor cells, on tumor vascularization as well as on the host’s immune cells, a combination of these effects contributing to the long-term control of the tumor [9]. The vascular modifications depend on PS, but in most of the cases lesions of the vascular

2

endothelium appear and thrombi form through the activation of the platelets and through the release of thromboxane. Also, vasoconstriction can occur due to the inhibition of formation/release of endothelial nitric oxide (NO) [10][11]. The inflammation is considered an important event in the development of the specific antitumor immunity; this type of therapy induces the expression of some cytokines and pro-inflammatory chemokines: interleukine (IL1ß, IL6), tumor necrosis alpha (TNFα), of inflammatory proteins of the macrophages (MIP-1, MIP-2) but also of the cellular adhesion molecules: Selectin E and intercellular adhesion molecule (ICAM). These modifications are accompanied by a massive infiltration of the treated tumor with leucocytes, most of them being neutrophils, mast cells and macrophages [9]. PHOTOSENSITIZERS

The ideal PS would be a chemically pure drug with preferential uptake in tumor, rapid clearance, and a strong absorption peak at light wavelengths >630 nm [2]. There are a few mechanisms which increase the retention of the PS in the tumors: tumor cells proliferate rapidly, have reduced lymphatic drainage, express more receptors for low density lipo-proteins, have low pH, have increased vascular permeability, have increased porphyrin binding collagen production, the macrophages in the tumor favor the captation of hydrophobic PS in cells [5]. FIRST GENERATION

The first generation of PS comprises hematoporphyrin (HpD), hematoporphyrin derivatives, and the purified commercial compound, Sodium Porfimer or Photofrin [2]. Photofrin, a mixture of porphyrin oligomers, has maximum absorption at 630 nm, a depth of action of 0.5 cm, has not specificity for tumor tissue, does not present systemic toxicity, and determines prolonged photosensitivity (4 to 8 weeks) [5]. It requires large dosages and a high fluence rate in order to obtain the same effects as the second generation of photosensitizing substances. SECOND GENERATION

The most utilized compound of the second generation is the 5-aminolevulinic acid (5-ALA)

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(Levulan), an intermediary product of the porphyrin biosynthesis, which, applied locally, increases the concentration of protoporphyrin IX (PpIX) in the tumor cells. The 5-ALA accumulation in the target cells is higher comparatively to the healthy tissue because these cells have an intense metabolic activity; they rapidly release the precursor, and because of a weak specificity of the ferrochelatase determine increased concentrations of the Pp IX [12]. Levulan only acts to a depth of 1.5 mm that is why it is used in the treatment of superficial cutaneous carcinoma, especially in the basocellular carcinoma and in the oral cavity dysplasias [13]. It does have advantages when compared to Sodium Porfimer because it persists less in the organism and the photosensitivity is reduced (1–2 days) [2]. Because of its hydrophylicity it enters the cells with difficulty. A few ALA alkyl esters with increased penetration in tissues were tried. The methyl ester ALA (Metvix) was approved by the European Union (EU) in 2001 for the treatment of the actinic keratoses and of the basal cells carcinoma [14]. Other porphyrins were also tested such as meso-tetrahydroxy tetraphenyl chlorin (m–THPC) or Foscan, which was approved by the EU in 2001 for the palliative treatment of head and neck cancers. It is more potent than Sodium Porfimer and ALA, it requires a small dosage for the control of the tumors (0.1mg/kg) and a fluence rate of 10–20J/cm2 and the photosensitivity persists for 2–4 weeks. It can penetrate deeper in the tissues because it has maximum absorption at 652mm. THIRD GENERATION

The new, 3rd generation PS are still in study, especially those activated by the long wavelength light which cause short photosensitivity and have broad tumor specificity. Although new classes of PS have entered in clinical trials few results have been published. These are: ethyletiopurpurin (SnET2), mono-L-aspartyl-chlorin e6 (Npe6), benzoporphyrin derivatives (BPD–Verteporfin), lutetium texaphyrin (Lu-Tex), phthalocyanines, anthracenes, rostaporfin (Photrex), purpurines, hypocrelines and hyperricin [5]. LIGHT SOURCES

The light sources used can be conventional, incoherent, cheap (halogen lamps, tungsten lamps, xenon lamps, fluorescent lamps), as well as unconventional, i.e. LASER. The conventional ones

have the advantage of being simple, cost effective, and can be applied both in vitro studies as well as in preclinical studies, in vivo. The disadvantages of this type of illumination are: significant thermal discomfort, low light intensity, and difficulties controlling the light dosage. Light emitting diodes (LED) represent a new light source for PDT. They can generate high luminous energy at the wished wavelength, and can be assembled in different shapes and sizes. The optimal illumination is obtained from LASERs because they are monochrome, have high intensity, and they can be easily coupled with fiber optics for endoscopic utilization or interstitial implants. The LASERs emit light with precise wavelengths but are expensive and require high-grade technical assistance [2]. PDT AS A CLINICAL APPLICATION FOR CANCER

For PDT, as for any new cancer therapy, it is important to identify the specific indications for the treatment and to evaluate its benefits and disadvantages relative to standard therapies. Before discussing in detail the possibilities of usage for the PDT in different types of carcinoma it is worth noting a few general aspects regarding this therapy. PDT is a treatment that usually requires only one administration of the photosensitizing substance followed by a single irradiation at a certain time interval, unlike radiotherapy and chemotherapy, which require more sessions for 6–7 weeks. The surgical treatment, although comprised of a single procedure, requires anesthesia and hospitalization for a period of time. The cost/effectiveness ratio suggests that PDT is an efficient treatment method, rather cheap and which increases the life expectancy when compared to other palliative treatments in head, neck and esophagus cancers [2]. Another argument for this type of therapy is that it is applied locally, the necrosis seldom passes 10mm and because the low penetrability of light, the healthy tissue around the tumor is not affected. The local side effects are minor; the functionality is kept with good tissue regeneration because the subepithelial collagen and elastin are kept intact. In the case of recurrence the treatment can be applied on exactly the same area that was previously irradiated which offers a real advantage compared to radiotherapy or surgery.

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In the case of early primary cancer and early relapses, PDT is indicated to patients with serious associated diseases and age-related disorders. It is performed according to a radical program when traditional therapeutic techniques (such as surgery and radiotherapy) are contraindicated. In the case of advanced tumors of tubular organs (such as the esophagus, cardiac portion of the stomach, trachea, rectum, as well as the main, intermediate, and lobe bronchi), PDT is performed to rechannel these organs. It is employed as a palliative therapeutic technique.

4

et al. have published extensively studies regarding their experience of using PDT in 103 patients, most of whom had high-grade dysplasia (HGD). The mean follow-up in this group was over 4 years. Of the 65 patients with HGD 78% had their HGD eliminated. On the basis of an intention-to-treat analysis, 54% had no residual BE [20]. The overall stricture rate for patients treated with PDT was 30%, but for those who required more than one PDT treatment it was 50%. Subsquamous, nondysplastic specialized intestinal metaplasia occurred in 4.9% of patients, but more importantly 3 patients (4.6%) developed subsquamous adenocarcinoma. CHOLANGIOCARCINOMA

ESOPHAGEAL CANCER

Due to very low survival rates at 5 years (12.5%) and also because the standard treatment, esophagectomy has a very high mortality, new treatments were employed, that are less invasive: endoscopic mucosal resection, coagulation and PDT [15]. For PDT, illumination is given using flexible cylindrical diffusers that are placed via an endoscope, near the tumor. The first studies with PDT in the esophagus were done as palliative treatment for obstructive tumors [16]. In 1995 Lightdale et al. published the result of a prospective randomized multicenter trial that compared Photofrin-PDT with Nd:YAG thermal ablation for the palliation of partially obstructing esophageal tumors. In this trial PDT proved its efficiency leading to the relief of dysphasia for a longer period of time compared to thermal ablation. Moreover, there were less treatment sessions needed and 9 out of 236 had a complete response (CR) after PDT, there was only one patient with esophageal perforation compared to 7% registered using thermal ablation [17]. PDT is efficient as a curative treatment for small, superficial tumors. A CR of 87% after 6 months and of 25% after 5 years was obtained in a group of 123 patients treated with sodium Porfimer [18]. PDT has also been proposed for the treatment of Barrett’s esophagus (BE) with high-grade dysplasias to destroy an area of thin tissues spread eventually over a wide area instead of a mass of tissues [19]. Only PDT using Photofrin® is approved by the FDA for the treatment of precancerous lesions in BE. Overholt

PDT can be used in the endoscopic palliative treatment of cholangiocarcinoma. Two smallrandomized studies have reported both palliative effects and an increase in median survival. For example, Ortner et al. conducted a trial of 39 patients with nonresecable cholangiocarcinoma who were randomized to receive either endoscopic stenting alone or in conjunction with PDT. The median survival of the 20 patients in the PDT group was 493 days compared to 98 days in the 19 patients who underwent stenting alone. The trial was terminated prematurely due to the favorable results [21]. Preliminary studies suggest that operative PDT might also improve survival for those patients undergoing surgical resection [22]. Currently the National Comprehensive Cancer practice guidelines for the treatment of hepatobiliary cancer do not list photodynamic therapy as one of the treatment options. STOMACH CANCER

Most of the reports of PDT for gastric cancer are included in large reports of PDT for various upper gastrointestinal cancers such as esophageal, cardia region and gastric [23]. The data currently available for gastric cancer are very similar to those for early esophageal cancer in that they involve small series of patients with short follow-up, a variety of tumor stages, different light dosimetry, and different means of evaluating tumor response [23][24]. If light delivery can be improved and if gastric tumors can be detected early enough, then PDT may play a role in their treatment.

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Photodynamic therapy in malignant treatment COLORECTAL CANCER

Only a handful of papers describe PDT for colon and rectal cancer; this is surprising when one considers the incidence of this disease [25][26]. PDT has been performed on smaller lesions, namely adenomas. The first study included eight patients with nine sessile villous adenomas, all but one of which recurred after Nd:YAG thermal ablation. After a follow-up period of 9 to 56 months, a biopsy was performed, and it was found that seven of the nine adenomas had been completely eradicated [27]. Use of the Nd: YAG laser to ablate adenomas is an alternative in nonoperative candidates. Although it successfully removes the lesion in 84% to 93% of cases with low morbidity and mortality rates (1% to 5% and 0%, respectively), the progression to invasive cancer after treatment is 5.7% to 9.1% [23]. Another study involved the treatment of large recurrent intestinal polyps in six patients with familial adenomatous polyposis. Of the two patients with colonic polyps, one was found to show a CR to treatment and no complications were reported [27]. HEAD AND NECK CANCER

Early-stage carcinomas in the head and neck area are normally treated with surgery and/or radiotherapy, while, for advanced disease, chemoradiation is standard treatment. Cure rates are good, especially for early disease, but can be associated with high morbidity (functional damage, swallowing, speech difficulties, xerostomia, trismus, and even osteonecrosis). PDT is effective for small superficial tumors or palliative treatment of recurrent disease but has the advantage of sparing tissue beneath the tumor, giving excellent long-term functional and cosmetic results [28]. Foscan is so far the most potent licensed photosensitizer for PDT. A large-scale multi-center study using Foscan-PDT for early stage (T1 and early T2) oral cancers has been done. The CR at 2 years was 75% after one PDT treatment but if re-PDT, surgery or radiotherapy were used a CR could be achieved in 8/13 patients [29]. PDT with mTHPC of second and multiple primary cancers resulted in CR rates of 67% for all tumors and 85% for T1 tumors [2]. For superficial tumors of the larynx and oropharynx similar results were obtained with a single treatment [30]. Of

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19 patients treated had a histological CR over a follow-up period of 13–71 months. Because the action of PDT is limited to small superficial tumors to treat deep-seated, bulky tumors, Lou et al. implemented interstitial PDT. The overall median survival of the patients was 14 months while 72% overall palliative benefit was achieved. The local control rate at 12 months was 41% [31]. BASAL CELL CARCINOMA (BCC)

Skin cancers are ideal for the study of the effects of PDT [2]. Dougherty was the first to treat three BCC of face with PDT in a 72 years old patient with PDT with a CR of over 85% [9]. Feyh et al. [30] in a study conducted on 57 patients using hematoporfirin 2 mg/kg and laser argon light at a fluence of 100 J/cm2 evaluated the results using skin biopsies after 2 months and saw a complete response in 51 patients. The patients that did not have a complete response after the first treatment were treated again with PDT and a CR was registered in all the patients. For the patients with only few localized lesions is used topical ALA or an ester of ALA called Metvix. ALA can be applied topical a few hours before irradiation with excellent results in the treatment of basal cell carcinoma. Soler et al. [32] studied the long-term effects of methyl 5-aminolaevulinate (MAL)-PDT in 59 patients with 350 BCC. Nodular tumors were curetted before PDT and MAL (160 mg/g) was applied to all tumors for 24 h or 3h prior to irradiation with a broadband halogen light source (50–200 J/cm2). Patients were followed for 2–4 years (mean 35 months). Overall cure rate was 79%, cosmetic outcome was excellent or good in 98% of the completely responding lesions. Svanberg et al. [33] showed an improved response after PDT in nodular BCC (from 64% up to 100%) if prior to the treatment drugs that enhance the penetration into the tissue are applied (dimethyl sulfoxide 20% and EDTA 4% added to ALA 20%). In a recent European multicenter, open, randomized trial, MAL-PDT for nodular BCC was compared with surgery. A total of 101 patients were included and received either PDT twice, 7 days apart (75 J/cm2 red light) or surgical excision. The primary end point of this trial was the clinically assessed lesion clearance at 3 months after treatment, besides cosmesis. The 3-month cure rate was similar with MAL-PDT or surgery (915 vs. 98%), the 24 month recurrence rate was

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10% with MAL-PDT and 25 with surgery. The cosmetic result was rated good/excellent in 85% of the patients receiving PDT vs. 33% with surgery [34]. A recent prospective phase III trial that compared the effects of ALA-PDT with cryotherapy in the treatment of superficial and nodular BCC did not find any difference between the two, only superior results for PDT regarding cosmetical results and quicker healing [35]. Complete resolution of 96% after 12 months for BCC is possible if the treatment is repeated after 7 days [36]. The guideline issued by the British Association of Dermatologists in 1999 regarding the treatment of BCC advises the use of PDT as the primary treatment for superficial BCC in spread forms or in those with multiple localizations. BOWEN’S DISEASE

Bowen’s disease is the affection with the best response to PDT. Topical PDT using 20% ALA has been extensively assessed in Bowen’s disease with more than 14 open and three randomized comparison studies [36]. In a recent study by Salim et al. ALA-PDT was compared to topical 5-fluorouracil (5-FU). In this bi-center, randomized, phase III trial, 40 patients with one to three lesions of previously untreated, histologically proven Bowen’s disease received either PDT (20% ALA in an o/w emulsion 4h prior to illumination) or 5-FU (once daily in week one and then twice daily during weeks 2–4). Twenty-nine of 33 lesions (88%) treated with pDT showed CR vs. 67% after 5-FU [37]. The data in the literature are controversial, though, as to the antitumor response to ALA-PDT. Some researchers give results of 90–100% while others only 50% [38]. BLADDER CANCER

The first license for Photofrin was granted in Canada in 1993 for use following transurethral resection for papillary tumors [14]. In clinical trials with HpD or Porfimer sodium PDT it was observed that PDT is more efficient in the treatment of superficial recurrent bladder carcinoma, with very high initial response rates (70–100% at 3 months) and long term response rates of 30–60%, compared to the responses after transurethral resection or treatment with bacillus Calmette-Guerin [39]. For whole–bladder PDT there was, however, a very high incidence of side effects: urinary frequency,

6

pain, and persistent reduction in bladder capacity. These complications were associated with excessive light doses and no uniform light delivery. Because of severe and long lasting side effects, Nseyo et al. suggested multiple treatments at lower drug dose [40]. Whole bladder PDT with green light and proper dosimetry remains an attractive treatment option for carcinoma in situ although this has not been fully evaluated. Recently, it was shown that ALA PDT, used solely or in association with mitomycin C, determines a complete response in 40%–52% of the cases at 18–24 months, without any significant side effects [41]. Kreigmair et al. used intravesical ALA in 10 patients with refractory superficial transitional cell carcinoma and obtained a complete remission in 4 patients after 10–12 weeks and a partial one in 2 patients. Fifty percent of patients had progressive disease that required cystectomy after a mean follow-up of 15 months. It has been shown that with repeated PDT treatments, it is possible to limit or inhibit progression of disease and cures a proportion of patients [42]. PROSTATE CANCER

Since the beginning of the 90’s there were attempts to use Foscan and ALA PDT to treat patients with prostate cancer that do not respond to radiotherapy. In studies using canine models it was shown that PDT leads to necrosis of the prostatic tissue and does not affect the collagen. This suggests that using a proper dosage of light and PS you can destroy the whole prostatic tissue and not affect the perineal anatomy [43]. There are first phase trials using mTHPC in the treatment of recurrent forms of cancer [2]. Also Motexafin lutetium and Tookad were used for the complete ablation of the gland. This therapy involves the implantation of multiple diffuser fibers into the prostate gland through a transperineal brachytherapy template. Because there is a difference in the optical properties of the different tissues in the prostate we ought to measure in real time the concentration of PS and light fluence. Protection of the pelvic nerve also becomes an inevitable challenge during total ablation because we need to minimize the adverse effects on sexual and urinary functions [22]. Using Pd-bacteriopheophorbide or Tookad, also known as WST09 (NegmaLerads/Steba Biotech), has some advantages: rapid clearance,

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profound penetration of the light leading to important destructions in the glandular tissue; it affects primarily the blood vessels that feed the tumor. This supports the approach being used in current Phase I/II clinical trials of Tookad-PDT for recurrent prostate cancer [22]. LUNG CANCER

Many publications have shown the therapeutic usefulness of PDT in different stages of endobronchial disease. Palliative treatment of obstructive cancer with HpD or Porfimer sodium PDT was safe and resulted in symptom relief in almost all patients [44]. The effects are similar to those obtained with Nd:YAG laser but they last longer [14]. The results of a multicentric trial which compares the two modalities reported, but not published, shows a more durable response with PDT and the superiority of PDT comparatively to Nd: YAG for relief of dyspnea, cough and haemoptysis. PDT has also been used as a curative treatment in early lung cancer. Overall, 5-year survival rates were in the range of 56%–70% with a disease specific 5-years survival rate of 90% for carcinoma in situ. The optimum response appeared to be in patients with small tumors less than 1 cm

in length (97% versus 42.9% for tumors larger than 1 cm) [46]. Another indication for endobronchial PDT is field cancerization or recurrence of tumors after resection or irradiation. Malignant pleural mesothelioma, often related to asbestos exposure, responds poorly to conventional therapies and it is very aggressive. Photofrin-PDT has been tested as an adjuvant intraoperative modality in several countries and proved to be safe and efficient in stages II mesothelioma and I. To improve its efficiency it should be tried in combination with hyperbaric oxygenation [22].

CONCLUSIONS

In conclusion, future research will undoubtedly be directed toward the development of improved photosensitizers increased tumor normal selectivity and fewer side effects. The research is also focusing on more efficient light delivery and increased understanding of the optical properties of tissues in addition to the effects of drug and light fractionation. Only when all these issues have been addressed will PDT fully realize its potential role as a major form of cancer treatment.

Terapia fotodinamică (PDT) este o tehnică nouă utilizată în tratamentul neoplaziilor solide şi se bazează pe formarea de singlet oxigen indusă de fotosensibilizatori după iradierea cu lumină vizibilă. În articol se discută mecanismul interacţiunii fotosensibilizant – lumina precum şi efectele produse în tesutul ţintă. PDT a fost aprobată în multe ţări în tratamentul cancerelor din sfera ORL, a celor de esofag, plămân, vezică urinară, piele. Efectele antitumorale ale PDT se datorează atât actiunii citotoxice directe asupra celulelor tumorale cât şi leziunilor vasculare care apar şi activării imunităţii gazdei. Se discută în articol indicaţiile clinice ale PDT şi se trec în revistă principalele clase de fotosensibilizanţi. În ciuda rezultatelor promiţătoare obţinute în primele trialuri clinice sunt necesare în continuare studii pentru a transforma această modalitate de tratament într-o tehnică modernă. Corresponding author: Adriana Filip, MD Physiology Depart., “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca E-mail:[email protected]

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Nutrigenomics/Nutrigenetics NICOLETA MITROI1, MARIA MOŢA2 1

Clinical County Emergency Hospital Craiova, Clinic of Diabetes Nutrition and Metabolic Diseases 2 University of Medicine and Pharmacy Craiova, Diabetes Department

Since Hippocrates it is known that nutrition plays a very important role in maintaining health, people being advised to consider “food intake as a real medicine” [1][2]. Modern science shows that important and necessary for an optimal state of health is not only the intake of some specific nutrients, but, above all, specific quantities of each and every nutrient which are to be taken. New notions have consequently appeared, such as dietetic recommendations and nutritional epidemiology. At the same time, it has been emphasized that nutrition can directly contribute to diseases appearance (nutrients/food generally interact with the genes in a “benign” way, but in some circumstances this interaction can also have fatal consequences) [1–3]. Human development is influenced by both environmental factors (diet, smoking, education, physical activity etc.) and heredity; both factors should be given equal attention, if our aim is to maintain the state of health. Experimental studies are often dedicated either only to the influence of environmental factors or exclusively to genes’ influence and not to both simultaneously [4]. Modern techniques and methods of study are designed to solve the problem. Key words: nutrients, gene, epigenetic interactions, genetic variations.

Last generation techniques allow the study of all genes, proteins and metabolites in a given sample, enabling at the same time the correlation of information. The result of these investigations consists in creating new terms which are characterized by the fact that they all contain the Greek suffix “ome”/”omic” (in translation complete, global, totally or whole): Genomics=global genes’ analysis; Transcriptomics=global analysis of mRNA; Proteomics=proteins’global analysis; Metabolomics= metabolites’global analysis [5]. Nutrigenomics studies the interaction between nutrients and genes [1][6]. Nutrients modulate the expression and regulation of coding genes of proteins, metabolisms, cellular differentiation and growth [1, 6]. Nutrigenomics has a double role: on the one hand, to explain the functional interaction between food bioactive components and human genome (at molecular, cellular and systemic level); on the other hand, the role of Nutrigenomics is to reveal the effects of genic variation upon dietdisease interaction. The study focused on the discovery of the specific response of each individual to food items, aiming to develop individualized dietetic recommendations [1][7]. At present, two terms are used to define the two scientific aspects: Nutrigenomics and Nutrigenetics. Although the two terms are intimately ROM. J. INTERN. MED., 2008, 46, 4, 295–304

related, they need nevertheless a fundamental different approach in understanding gene-diet relation. On the long term, the two scientific domains interact and share a common goal: to improve the state of health and to prevent the appearance of nutritional diseases (Fig. 1).

Fig. 1. – Personalized nutrition (from MUTCH D.M., WAHLI W., WILLIAMSON G., Nutrigenomics and nutrigenetics: the emerging faces of nutrition. The FASEB Journal, 2005, 19: 1602–1616, modified).

DIET-DISEASE RELATION

The understanding of diseases’ appearance has considerably progressed since human genome sequencing has been performed for some eukaryotes

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[8]. From about 1000 genes that have been by now associated with the development of some diseases, 97% are related to diseases with monogenic determinism [9]. Modifying the intake of some food items or nutrients, one can prevent diseases with monogenic determinism, for example galactosemia and phenylketonuria. On the other hand, the diseases which tend to become epidemical, such as neoplasy, obesity, diabetes and cardiovascular diseases appear most frequently as a result of the disfunctionality of more genes, being thus called polygenic diseases. If we consider establishing some dietetic solutions in order to prevent these diseases, then we must know not only the way in which a nutrient can influence a biologic system, but, above all, the way in which a complex ingredients mixture – which is actually the diet – interacts in modulating biologic functions [10]. FOOD/NUTRIENT – GENE INTERACTIONS

Food contains genic expression regulators, such as: nutrients: fatty acids, selenium, zinc; non-

nutrients: phytochemicals, isoflavones; metabolites of food components: eicosanoides; compounds resulted from food processing: heterocyclic amines; compounds resulted from intestinal metabolism under the influence of microbial flora [4]. Nutrient – gene relation is characterized by the existence of the following interaction types: −

direct interactions: the nutrients, possibly as a result of the interaction with a receptor, behave as transcription factors which can bind at the DNA level, causing the acute expression of a gene. − epigenetic interactions: the nutrients can modify DNA structure, case in which the expression alteration of that particular gene becomes chronic. − genetic variations: common genetic variations (single nucleotide polymorphism-SNPs) can modify a gene’s expression or function. The interaction between nutrient and gene can cause the disturbance of the following processes (Fig. 2): genic transcription; mRNA translation; mRNA processing; mRNA determining; post translational modifications.

Food:

Genic transcription

Direct interactions

-nutrients -non-nutrients

mRNA translation

-metabolites of food components -compounds resulted from intestinal metabolism

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GENE

mRNA processing mRNA determining

Epigenetic interactions

Post-translational modifications

Genetic variation

SNPs

Fig. 2. – The complex relationship between nutrition and genic expression.

NUTRIGENOMICS

Nutrition can no longer be considered to be just the science of simple epidemiologic studies which aim to discover the relationships between nutrition and chronic diseases in genetically uncharacterized populations. At the same time, one must also take into consideration the integration of cellular biology, biochemistry and genetics knowledge. Nutrigenomics aims to identify the biological markers which can describe more accurately the state of health (by discovering new molecular targets).

From the three possible interactions between a nutrient and a gene (mentioned above), Nutrigenomics evaluates the acute effects of the nutrients upon the transcription process and epigenetic interactions. ACUTE EFFECTS OF THE NUTRIENTS UPON GENES TRANSCRIPTION [11]

There are several nutrients which can modify genes expression, acting as transcription factors: − vitamin A interacts with retinoic acid receptor, and the resulted complex causes transcription

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activation or inhibition, in the case of attaching to a certain level of gene promotor region [12] − fatty acids can interact with PPAR (peroxisome proliferator-activated receptors), which can modify genes expression by attaching at DNA level [13] − the interaction between vitamin D and its receptor − Calcium-calcineurin interaction − Zinc – transcription factor 1 interaction (metalresponsive transcription factor 1) [14]. All these examples refer to a nutrient which acts like a short term trigger and causes the acute modification of the transcription process; this effect generally disappears once the exposure to the specific nutrient has been stopped. EPIGENETIC INTERACTIONS

Epigenetic mechanisms have been recently implicated in describing the way in which nutrients modify genes expression; these are mediated either by DNA methylation or by histone methylation or acetylation, or by both [15]. Such epigenetic modifications can lead to genic expression perturbance which can last the whole life and can be transmitted to the coming generations. DNA methylation is usually performed at the level of cytosine base (regions 5’-CpG-3’) [16], influencing thus both genes transcription process and genome stability [17]. If this modification takes place at the level of promotor region, the result is a perturbance in genic expression [18]. Normally, the intensification of methylation process associates with genic expression reduction [18] because the methylated regions 5’-CpG-3’ possess the property to provoke protein attachment, hindering thus the access of transcription factors [19]. Once the methylation is completed, it will replicate each time when the gene is copied. DNA is surrounded by proteins (histone) which hinder the access to genes promotor regions. If histone methylation or acetylation take place [20], then some channels can be created, through which transcription factors can penetrate, causing promoters activation. Diet modification concerning methyl groups can induce stable modifications in genes methylation, disturbing genic expression and resultant phenotype [21]. For example, the choline quantity (methyl donor) that is to be found in the nutrition of

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pregnant mice females influences proliferation and apoptosis rates of nervous cells in the fetus brain [22]. Colin quantity reduction in food was associated with DNA methylation perturbance [23]. Differences regarding visual, spatial and auditive memory have been also observed [24]. If colin intake was high, the offspring had a 30% higher visual, spatial and auditive memory [25], this high colin exposure during intrauterine period causing also the perseveration of memory capacity. Epigenetic effects of nutritional modifications were also observed in humans. In this sense, the specialists discovered that persons whose paternal grandparents were confronted in the growing up period with the absence of some nutrients from the diet (they did not have a diverse diet) presented a higher life expectation and a risk of diabetes mellitus with up to 75% lower [26]. Pembrey [27] demonstrated that these nutrition effects are being performed through epigenetic mechanisms hereditary transmitted. In a similar way, epigenetic mechanisms can be involved in carcinogenesis process modulation in adults. LONG-CHAIN POLYUNSATURATED FATTY ACIDS (LC-PUFA) AND CARCINOGENESIS

LC-PUFA are implicated in numerous processes such as: growing process, neurological development, the development of muscular and adipose mass, reproduction, innate and acquired immunity, virus infections, bacteria and parasites, the incidence and gravity of some diseases, including neoplasias, atherosclerosis, stroke, arthritis, diabetes, osteoporosis, neurodegenerative and inflammatory diseases and skin pathology [28][29]. The roles played by LC-PUFA are associated to their capacity to act (or to be transformed in) as ligands for various transcription factors, including PPAR, hepatic nuclear factor 4, the protein responsible for sterol transport, liver-Xreceptor alpha, nuclear factor κ β [30][31]. It was also demonstrated that fatty acids are involved in lipid metabolism modifications (related to phosphorlipids, triglycerides and cholesterol esters) [32][33]. LC-PUFA represents also the most frequently studied lipid fraction regarding its role in neoplasias appearance [34]. It has been demonstrated that fish oil (rich in omega-3 fatty acids) inhibits the cell growth of a colon tumor, both in vitro and in vivo [35]. Preliminary results indicate the involvement of these nutrients in function modulation of some

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transcription factors, RNA transcription process, prostaglandins synthesis, genes activity responsible for nitric oxide synthesis [36][37]. SITOSTEROLEMIA

Sitosterolemia is a rare autosomal recessively inherited metabolic disorder, characterized by hyperabsorption and decreased biliary excretion of dietary sterols, leading to hypercholesterolemia, xanthomatosis, premature development of atherosclerosis, and abnormal hematological and liver function test results. Sitosterolemia shares several clinical characteristics with familial hypercholesterolemia; however, in contrast to familial hypercholesterolemia, it presents normal to moderately elevated total sterol levels and very high levels of plant sterols (10–25 times). The results of the Berge’study explained two disputed aspects: the mechanism through which plant sterols – structurally similar to cholesterol – are normally absorbed in a negligible proportion and the fact that patients with sitosterolemia show an increased absorption of plant sterols [38]. Study design: evaluation of mice subjected to the treatment with a drug that interferes with lipid metabolism; mRNA profiles for different tissues were compared with those of normal mice. It was discovered a gene belonging to the family “ATP binding cassette” (ABC), which includes genes that codify the proteins responsible for lipid transport. Berge concluded that the proteins codified by genes belonging to this family, respectively ABCG5 and ABCG8, are the ones implicated in the transport modulation of animal or vegetal sterols absorbed at intestinal level. The human homologue of this gene was then identified, and, after its cloning, it was used in the screening of patients with sitosterolemia. All evaluated cases proved to carry mutations of this gene, lacking thus the control mechanism responsible for a selective and controlled cholesterol transport. This fact leads to increased sterol absorption, including those of vegetal origin. NUTRIGENETICS

Nutrigenetics studies the way in which the genetic inheritance of each individual interferes in the response of the organism to nutrition. There are more than 10 million SNPs (Single Nucleotide Polymorphisms) which appear in more than 1% of

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the population [39], some of them being encountered with a frequency which varies between 5% and 50%. Most humans are heterozygote for ≥ 50.000 SNPs [40]. The SNP Consortium is in charge with mapping important polymorphic sites which dictate phenotype differences. The largest SNP database contains approximately 7.7 million SNPs (dbSNP). Some of these cause either genic expression perturbance, or structural and functional modifications of gene codified proteins. There are some genes (so-called “susceptible”) which are characterized by the existence of a specific interrelation with diet [41]. In order to identify these genes, one must take into account several conditions: genes chronically activated in illness periods and which proved to be sensitive to dietary interventions; genes with important functional variations; genes that are important in biological cascades; polymorphisms with high prevalence in population; genes with associated biomarkers, enabling thus the performing of clinic studies. In some regions of the genes (≤ 500kb) we can encounter combinations of some SNPs related to one another which are identified in many persons (haplotype blocks). These associated SNPs combinations are transmitted over several generations [42]. The identification of some alleles of a haplotype block can lead to the description of all polymorphic sites from this region characteristic for a group of people with the same race or ethnical background, and this could make SNPs analysis much more accessible for nutritionists and clinicians. GENETIC VARIABILITY CAN INFLUENCE DIETARY NECESSITIES

We mention here some examples of SNPs which are known to influence food intake necessities of a nutrient: −

5,10 methylentetrahydrofolate reductase (MTHFR) is an enzyme implicated in folate metabolism. The gene which codifies for MTHFR has a common SNPs (allele C677T) that is associated with the reduction of enzymatic activity translated in the reduction of homocystein conversion in methionine. Homozygote subjects are characterized by high plasmatic homocystein concentrations [43] (considered to be cardiovascular risk factor) [44]. Although the nutrient-gene interaction has been described

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[45], there are still controversies regarding the fact that diet supplementation with folate could reduce cardiovascular risk [46]. According to some studies, the frequency of these SNPs in the population is 10–15% [47], and 15–30%, according to others [43]. − there are SNPs which modify the risk of appearance of organ dysfunction [81, 82, 83, 84]. For example, in case of a low choline food intake, some persons develop hepatic and muscular affections, others do not. Premenopausal women carrying very frequent SNPs (MTHFD1G1958A) show a 15 times higher risk of developing choline deficiency compared to those who do not have these SNPs [85]. It has been also observed that the appearance risk of neural tube defects in offspring is increased also if mothers carry these SNPs [86] and if they had a diet characterized by a low choline intake [51]. It is interesting to find out if these women (with the higher risk) are also SNP G1958A carriers. − PEMT gene (phosphatidylethanolamine n-methyltransferase) codifies a protein responsible for choline endogeneous formation at hepatic level [52] and it is induced by estrogen [53]. Studying organ dysfunctions which can appear in case of choline deficit, one discovered that a SNPs present at the level of PEMT gene promotor region (rs12325817) is associated with susceptibility increment only in women [51]. − SNPs of SREBP gene (sterol response element binding protein) influence the hepatic lipogenesis process induced by fructose [54]. − the gene for PPAR-alpha has a SNPs which was associated with the modifications of total cholesterol, LDL cholesterol and apolipoprotein B concentrations [55], perturbing the organism response to n-6 polyunsaturated fatty acids intake. In persons carrying the variant allele, the high n-6 polyunsaturated fatty acids intake was associated with a significant reduction of triacylglycerol concentration [55]. Several studies evaluated the correlations between some SNPs and particular phenotypes, aiming to investigate the relations between the millions of sites of recently discovered genetic variants. INSULIN RESISTANCE AND TYPE 2 DIABETES MELLITUS

Some observational studies described a higher type 2 DM incidence in persons with low

physical activity or high food intake [56]. Once the results of the first study carried out in this direction had been presented (West and Kalbfleish [57]), it was known that a diet characterized by a high caloric and lipid intake plays an important role in type 2 DM appearance. According to present information, both genetic and environmental factors are significant for diabetic disease etiology. Recent studies on mice carrying genetic variations which interfere with lipid metabolism have proved the relationship between lipids and diabetes [58]. The evaluation of genes implicated in fatty acids and triglycerides synthesis played an important role in demonstrating that neither diabetes, nor insulin resistance are diseases with monogenic determinism; numerous genes implicated in lipid metabolism regulation and insulin sensitivity modulate DM appearance risk (for example the genes for adiponectin and resistin) [59]. The present data also show that these genes are diet responsive, demonstrating at the same time the existence of correlations between genotype and diet in developing type 2 DM predisposition. CORONARY HEART DISEASE

Epidemiologic studies showed the existence of a correlation between lipids and coronary heart disease [60], and The National Cholesterol Education Program added that two of the lipoprotein fractions are the main targets in fighting this disease: LDL and HDL. In dyslipidemia treatment, the optimization of lifestyle represents the first therapeutic step and includes diet and physical activity modifications, combined with the determination to reduce smoking and lose weight. Medicamentous treatment is the second step of the therapy; statines are here recommended, because they inhibit the activity of hepatic 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), causing thus the reduction of sanguine cholesterol level [61]. But we must also take into consideration the fact that there are individual differences in treatment response [100], these being associated with genetic differences [62]. Some susceptible genes have been discovered by now and their SNPs have been evaluated [63]. These genes codify: cholesterol ester transfer protein (CETP); lipoprotein lipase (LPL); hepatic triglyceride lipase (HL); LDLreceptor; apolipoprotein E(APOE); apolipoprotein A1 (APOA1) [64]; ATP binding cassette transporter A1 (ABCA1); lecithin-cholesterol acyltransferase (LCAT).

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Nutrigenetics studies reported the fact that some of the mentioned genes and their variants (for example APOA1 and LPL) are susceptible to respond to diet modifications. LIPOPROTEIN LIPASE (LPL)

Abnormalities in LPL function have been found to be associated with a number of diseases (atherosclerosis, obesity, Alzheimer’s disease and dyslipidaemia associated with diabetes) [65]. There have been described some polymorphisms characterized by LPL dysfunction that contribute to an early appearance of coronary heart disease, mainly because they associate with high triglycerides levels [66]. Merkel evaluated some of the most common LPL polymorphisms and showed that these cause modifications of circulating lipids in pregnant women (who frequently present high levels of triglycerides and total cholesterol). Thus, although triglycerides remain unmodified, it has been shown that some of SNPs for LPL modify HDL level and pregnant women’s susceptibility for coronary heart disease [67]. Another LPL polymorphism was also studied (T495G HindIII), results indicating that this plays an important role in the appearance of coronary heart disease, obesity and diabetes [68]. The respective SNPs is characterized by high triglycerides and low HDL levels. Lipid profile perturbation and the diseases correlated with T495G HindIII proved to be influenced by several factors, such as physical activity [69] and low caloric intake [70]. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARS)

PPARs mediates the cellular response to different biologic and synthesis factors, such as fibrates, thiazolidinediones, fatty acids and their derivatives [71]. Three types of PPAR have been identified: α, β/δ and γ [72]. PPAR-γ activation promotes adipogenesis and TG storage through its action on some genes, inclusively those responsible for fatty acids transport, including them in TG and through glyceroneogenesis [95]. PPAR- activation by thiazolidinediones will result in lowering the circulating fatty acids level by promoting their capture in adipocytes and, finally, by increasing insulin sensitivity. Several studies on mice have shown that PPAR-γ +/– mice possessed an insulin sensitivity superior to that expected by investigators

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[73]. It has also been discovered that the absence of specific hepatic PPAR-γ in an obese mouse (with leptin deficiency) is associated with the absence of lipid accumulation capacity in the liver, resulting in appearance of insulin-resistance and type 2 diabetes mellitus [74]. The important roles played by PPAR-γ showed that the genes for these can be involved in type 2 diabetes mellitus appearance [75]. It is demonstrated that a certain mutation of the gene for PPAR-γ2 (replacing proline with alanine) increases insulin sensitivity and reduces the risk of type 2 diabetes mellitus appearance [77]. Thus, both The Bogalusa Heart Study [136] and The Go-DARTS Study [135] have registered the association of the polymorphism of the gene for PPAR-γ2 (Pro12Ala) with insulin sensitivity increment and, respectively, with a reduction of heart attack risk associated to diabetes. Other trials have reported the fact that Pro12Ala is associated with a high body mass index, this being one of the factors which predispose to type 2 diabetes mellitus appearance [78]. Through the correlations established between genetic variability and diet, on the one hand, and insulin resistance, type 2 diabetes mellitus and cardiovascular diseases, on the other hand, these studies have demonstrated the way in which individual’s genotype determination can contribute to nutrition management in order to prevent disease appearance. CONCLUSIONS

All examples mentioned above show that it is possible today to understand both the way in which genes’ SNPs are implicated in modifying nutritional necessities, and how nutrition can influence genetic expression. In the future we may be able to identify thousands of SNPs and at least an equal number of epigenetic modifications. It must be also mentioned that these modifications in the genes can interact in a complex way.We must notice that the integrated analysis of genetic, proteomic, metabolomic characteristics and of the complex interaction between genome and food/nutrient (nutritional phenotype) should represent the evaluation basis of the nutritional status of a person. It becomes thus obvious that there is a need for collective effort of the scientific community (genetics, molecular biology, biochemistry, nutrition, bioinformatics etc.). For these reasons, nutrition represents in this

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century an exciting field of research, new information being obtained almost every day regarding the way in which the interaction between lifestyle and genotype contributes to the preservation of

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the state of health or, on the contrary, to disease appearance. Each such piece of information helps us to be one step closer to what we call “personalized nutrition.”

Nutrigenomica studiază interacţiunea dintre nutrienţi şi gene. Genele influenţează modul de metabolizare al nutrienţilor şi, reciproc, nutrienţii modulează expresia şi reglarea genelor codante ale proteinelor, metabolismelor, diferenţierii şi creşterii celulare. Astfel, rolul nutrigenomicii este dublu: pe de o parte, scopul este de a înţelege interacţiunea funcţională dintre componentele bioactive ale alimentelor şi genomul uman (la nivel molecular, celular şi sistemic). Pe de altă parte, rolul nutrigenomicii este de a înţelege efectele variaţiei genice asupra interacţiunii dietă-boală. Cercetarea s-a concentrat în acest sens pe descoperirea răspunsului specific fiecărui individ la alimente, urmărind crearea unor recomandări dietetice individualizate. Sunt folosiţi în prezent doi termeni pentru a defini cele două laturi ştiinţifice: nutrigenomica şi nutrigenetica. Deşi cei doi termeni sunt intim asociaţi, totuşi ei presupun o abordare fundamental diferită în înţelegerea relaţiei gene-dietă. Ceea ce, pe termen lung, îşi propun drept ţintă este însă comun şi este reprezentat de: îmbunătăţirea stării de sănătate şi prevenirea apariţiei bolilor prin nutriţie, iar acest aspect a condus la intrepătrunderea celor două domenii ştiinţifice. Corresponding author: Maria Mota, Professor University of Medicine and Pharmacy Craiova, Diabetes Department 2–4 Petru Rareş Str., Craiova E-mail: [email protected]

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ORIGINAL ARTICLES

Impact of Highly Active Antiretroviral Therapy on Cytomegalovirus Viraemia in the Absence of Specific Anti-Cytomegalovirus Therapy RALUCA MIHĂILESCU1, VICTORIA ARAMĂ1*, SIMONA PARASCHIV1, A. STREINU-CERCEL1*, D. OŢELEA1, DANIELA MUNTEANU1, MIHAELA IOSIPENCO1, CARMEN CHIOTAN1, OTILIA ELISABETA BENEA1*, MARIANA MĂRDĂRESCU1, MIHAELA RĂDULESCU1, ADRIANA HRISTEA1*, RODICA UNGURIANU1, S.S. ARAMĂ*, ANCA STREINU CERCEL1*, RUXANDRA CĂLIN1*, C. BĂICUŞ* 1

“Prof. Dr. Matei Balş” National Institute of Infectious Diseases, Bucharest, Romania * “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

Objectives. 1) to evaluate the effect of HAART on CMV viraemia in co-infected patients, in the absence of specific anti-CMV therapy; 2) to compare 2 molecular biology techniques for the detection and quantification of CMV-DNA in these patients. Methods. We present the preliminary data of an ongoing prospective research grant on newly diagnosed HIV seropositives, in a tertiary care hospital, during June 2006- June 2008. Clinical, virological (HIV and CMV viraemia) and immunological (CD4) screening was performed every 3 months. The CMV viraemia was performed by RoboGene Human Cytomegalovirus Quantification kit (aj Roboscreen). We retested all undetectable CMV viremia found in patients with CD4