The TRAM flap for breast reconstruction : Studies on perioperative cutaneous blood flow, vasoconstriction, and indices of obesity

Breast reconstruction is performed for 10-15 % of women operated on for breast cancer. A popular method is the TRAM (transverse rectus abdominis musculocutaneous) flap formed of the patient’s own abdominal tissue, a part of one of the rectus abdominis muscles and a transverse skin-subcutis area over it. The flap can be raised as a pedicled or a free flap. The pedicled TRAM flap, based on its nondominant pedicle superior epigastric artery (SEA), is rotated to the chest so that blood flow through SEA continues. The free TRAM flap, based on its dominant pedicle deep inferior epigastric artery (DIEA), is detached from the abdomen, transferred to the chest, and DIEA and vein are anastomosed to vessels on the chest. Cutaneous necrosis is seen in 5–60 % of pedicled TRAM flaps and in 0–15 % of free TRAM flaps. This study was the first one to show with blood flow measurements that the cutaneous blood flow is more generous in free than in pedicled TRAM flaps. After this study the free TRAM flap has exceeded the pedicled flap in popularity as a breast reconstruction method, although the free flap it is technically a more demanding procedure than the pedicled TRAM flap. In pedicled flaps, a decrease in cutaneous blood flow was observed when DIEA was ligated. It seems that SEA cannot provide sufficient blood flow on the first postoperative days. The postoperative cutaneous blood flow in free TRAM flaps was more stable than in pedicled flaps. Development of cutaneous necrosis of pedicled TRAM flaps could be predicted based on intraoperative laser Doppler flowmetry (LDF) measurements. The LDF value on the contralateral skin of the flap decreased to 43 ± 7 % of the initial value after ligation of the DIEA in flaps developing cutaneous necrosis during the next week. Endothelin-1 (ET-1) is a powerful vasoconstrictory peptide secreted by vascular endothelial cells. A correlation was found between plasma ET-1 concentrations and peripheral vasoconstriction developing during and after breast reconstructions with a pedicled TRAM flap. ET-1 was not associated with the development of cutaneous necrosis. Felodipine, a vasodilating calcium channel antagonist, had no effect on plasma ET-1 concentrations, peripheral vasoconstriction or development of cutaneous necrosis in free TRAM flaps. Body mass index and thickness of abdominal were not associated with cutaneous necrosis in pedicled TRAM flaps.

Effects of opioids on ventilation and hemodynamics

Opioids are most commonly used for treatment of severe pain. However, the fear of respiratory depression has restricted the use of opioids. Depending on the monitoring system used, different modes of opioid respiratory effects have been noted in previous studies. All opioids also cause alterations in hemodynamics at least to some extent. The main goal of this series of investigations was to elucidate the native ventilatory and hemodynamic effects of different opioids. Studies I-IV each involved 8 healthy male volunteers. Study V involved 13 patients with lower or upper extremity traumas. The opioids studied were morphine, oxycodone, pethidine, fentanyl, alfentanil, tramadol and ketamine. The respiratory parameters used in this study were breathing pattern measured with respiratory inductive plethysmography, gas exchange measured with indirect calorimetry, blood gas analysis and pulse oximetry. Hemodynamics was measured with arterial blood pressure, heart rate and oxygen consumption. Plasma catecholamine and histamine concentrations were also determined. All opioids studied caused an alteration in respiratory function. Respiratory rate, alveolar ventilation and minute ventilation decreased, while tidal volume increased in most situations. Breathing pattern was also significantly affected after opioid administration. The respiratory depression caused by oxycodone was deeper than the one caused by same dose of morphine. An equianalgesic dose of tramadol caused markedly smaller respiratory depression compared to pethidine. The potency ratio for respiratory depression of fentanyl and alfentanil is similar to analgesic potency ratio studied elsewhere. Racemic ketamine attenuated the respiratory depression caused by fentanyl, if measured with minute ventilation. However, this effect was counteracted by increased oxygen consumption. Supplemental oxygen did not offer any benefits, nor did it cause any atelectasis when given to opioid treated trauma patients. Morphine caused a transient hemodynamic stimulation, which was accompanied by an increase in oxygen consumption. Oxycodone, alfentanil, fentanyl, tramadol and pethidine infusions had minimal effects on hemodynamics. Plasma catecholamine concentrations were increased after high dose opioid administration. Plasma histamine concentrations were not elevated after morphine nor oxycodone administration. Respiratory depression is a side effect noted with all opioids. The profile of this phenomenon is quite similar with different opioid-receptor agonists. The hemodynamic effects of opioids may vary depending on the opioid used, morphine causing a slight hemodynamic stimulation. However, all opioids studied could be considered hemodynamically stable.

Infections in lung and heart transplant recipients : Studies on the impact of bronchoscopy in the diagnosis of respiratory infections and detection of viral infections in blood and bronchoalveolar lavage fluid

Infection is a major cause of mortality and morbidity after thoracic organ transplantation. The aim of the present study was to evaluate the infectious complications after lung and heart transplantation, with a special emphasis on the usefulness of bronchoscopy and the demonstration of cytomegalovirus (CMV), human herpes virus (HHV)-6, and HHV-7. We reviewed all the consecutive bronchoscopies performed on heart transplant recipients (HTRs) from May 1988 to December 2001 (n = 44) and lung transplant recipients (LTRs) from February 1994 to November 2002 (n = 472). To compare different assays in the detection of CMV, a total of 21 thoracic organ transplant recipients were prospectively monitored by CMV pp65-antigenemia, DNAemia (PCR), and mRNAemia (NASBA) tests. The antigenemia test was the reference assay for therapeutic intervention. In addition to CMV antigenemia, 22 LTRs were monitored for HHV-6 and HHV-7 antigenemia. The diagnostic yield of the clinically indicated bronchoscopies was 41 % in the HTRs and 61 % in the LTRs. The utility of the bronchoscopy was highest from one to six months after transplantation. In contrast, the findings from the surveillance bronchoscopies performed on LTRs led to a change in the previous treatment in only 6 % of the cases. Pneumocystis carinii and CMV were the most commonly detected pathogens. Furthermore, 15 (65 %) of the P. carinii infections in the LTRs were detected during chemoprophylaxis. None of the complications of the bronchoscopies were fatal. Antigenemia, DNAemia, and mRNAemia were present in 98 %, 72 %, and 43 % of the CMV infections, respectively. The optimal DNAemia cut-off levels (sensitivity/specificity) were 400 (75.9/92.7 %), 850 (91.3/91.3 %), and 1250 (100/91.5 %) copies/ml for the antigenemia of 2, 5, and 10 pp65-positive leukocytes/50 000 leukocytes, respectively. The sensitivities of the NASBA were 25.9, 43.5, and 56.3 % in detecting the same cut-off levels. CMV DNAemia was detected in 93 % and mRNAemia in 61 % of the CMV antigenemias requiring antiviral therapy. HHV-6, HHV-7, and CMV antigenemia was detected in 20 (91 %), 11 (50 %), and 12 (55 %) of the 22 LTRs (median 16, 31, and 165 days), respectively. HHV-6 appeared in 15 (79 %), HHV-7 in seven (37 %), and CMV in one (7 %) of these patients during ganciclovir or valganciclovir prophylaxis. One case of pneumonitis and another of encephalitis were associated with HHV-6. In conclusion, bronchoscopy is a safe and useful diagnostic tool in LTRs and HTRs with a suspected respiratory infection, but the role of surveillance bronchoscopy in LTRs remains controversial. The PCR assay acts comparably with the antigenemia test in guiding the pre-emptive therapy against CMV when threshold levels of over 5 pp65-antigen positive leukocytes are used. In contrast, the low sensitivity of NASBA limits its usefulness. HHV-6 and HHV-7 activation is common after lung transplantation despite ganciclovir or valganciclovir prophylaxis, but clinical manifestations are infrequently linked to them.

Genistein and 17β-estradiol fatty acid esters in blood and adipose tissue and the structure-related antioxidant activity of estrogens on lipoproteins

Soy-derived phytoestrogen genistein and 17β-estradiol (E2), the principal endogenous estrogen in women, are also potent antioxidants protecting LDL and HDL lipoproteins against oxidation. This protection is enhanced by esterification with fatty acids, resulting in lipophilic molecules that accumulate in lipoproteins or fatty tissues. The aims were to investigate, whether genistein becomes esterified with fatty acids in human plasma accumulating in lipoproteins, and to develop a method for their quantitation; to study the antioxidant activity of different natural and synthetic estrogens in LDL and HDL; and to determine the E2 esters in visceral and subcutaneous fat in late pregnancy and in pre- and postmenopause. Human plasma was incubated with [3H]genistein and its esters were analyzed from lipoprotein fractions. Time-resolved fluoroimmunoassay (TR-FIA) was used to quantitate genistein esters in monkey plasma after subcutaneous and oral administration. The E2 esters in women s serum and adipose tissue were also quantitated using TR-FIA. The antioxidant activity of estrogen derivatives (n=43) on LDL and HDL was assessed by monitoring the copper induced formation of conjugated dienes. Human plasma was shown to produce lipoprotein-bound genistein fatty acid esters, providing a possible explanation for the previously reported increased oxidation resistance of LDL particles during intake of soybean phytoestrogens. Genistein esters were introduced into blood by subcutaneous administration. The antioxidant effect of estrogens on lipoproteins is highly structure-dependent. LDL and HDL were protected against oxidation by many unesterified, yet lipophilic derivatives. The strongest antioxidants had an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups. E2 ester levels were high during late pregnancy. The median concentration of E2 esters in pregnancy serum was 0.42 nmol/l (n=13) and in pre- (n=8) and postmenopause (n=6) 0.07 and 0.06 nmol/l, respectively. In pregnancy visceral fat the concentration of E2 esters was 4.24 nmol/l and in pre- and postmenopause 0.82 and 0.74 nmol/l. The results from subcutaneous fat were similar. In serum and fat during pregnancy, E2 esters constituted about 0.5 and 10% of the free E2. In non-pregnant women most of the E2 in fat was esterified (the ester/free ratio 150 - 490%). In postmenopause, E2 levels in fat highly exceeded those in serum, the majority being esterified. The pathways for fatty acid esterification of steroid hormones are found in organisms ranging from invertebrates to vertebrates. The evolutionary preservation and relative abundance of E2 esters, especially in fat tissue, suggest a biological function, most likely in providing a readily available source of E2. The body s own estrogen reservoir could be used as a source of E2 by pharmacologically regulating the E2 esterification or hydrolysis.

Cardiac function before and after treatment for various types of loading conditions and in myocardial restriction : A prospective study on pediatric patients with two- and three-dimensional echocardiography and measurement of serum natriuretic peptides

Background: The incidence of all forms of congenital heart defects is 0.75%. For patients with congenital heart defects, life-expectancy has improved with new treatment modalities. Structural heart defects may require surgical or catheter treatment which may be corrective or palliative. Even those with corrective therapy need regular follow-up due to residual lesions, late sequelae, and possible complications after interventions. Aims: The aim of this thesis was to evaluate cardiac function before and after treatment for volume overload of the right ventricle (RV) caused by atrial septal defect (ASD), volume overload of the left ventricle (LV) caused by patent ductus arteriosus (PDA), and pressure overload of the LV caused by coarctation of the aorta (CoA), and to evaluate cardiac function in patients with Mulibrey nanism. Methods: In Study I, of the 24 children with ASD, 7 underwent surgical correction and 17 percutaneous occlusion of ASD. Study II had 33 patients with PDA undergoing percutaneous occlusion. In Study III, 28 patients with CoA underwent either surgical correction or percutaneous balloon dilatation of CoA. Study IV comprised 26 children with Mulibrey nanism. A total of 76 healthy voluntary children were examined as a control group. In each study, controls were matched to patients. All patients and controls underwent clinical cardiovascular examinations, two-dimensional (2D) and three-dimensional (3D) echocardiographic examinations, and blood sampling for measurement of natriuretic peptides prior to the intervention and twice or three times thereafter. Control children were examined once by 2D and 3D echocardiography. M-mode echocardiography was performed from the parasternal long axis view directed by 2D echocardiography. The left atrium-to-aorta (LA/Ao) ratio was calculated as an index of LA size. The end-diastolic and end-systolic dimensions of LV as well as the end-diastolic thicknesses of the interventricular septum and LV posterior wall were measured. LV volumes, and the fractional shortening (FS) and ejection fraction (EF) as indices of contractility were then calculated, and the z scores of LV dimensions determined. Diastolic function of LV was estimated from the mitral inflow signal obtained by Doppler echocardiography. In three-dimensional echocardiography, time-volume curves were used to determine end-diastolic and end-systolic volumes, stroke volume, and EF. Diastolic and systolic function of LV was estimated from the calculated first derivatives of these curves. Results: (I): In all children with ASD, during the one-year follow-up, the z score of the RV end-diastolic diameter decreased and that of LV increased. However, dilatation of RV did not resolve entirely during the follow-up in either treatment group. In addition, the size of LV increased more slowly in the surgical subgroup but reached control levels in both groups. Concentrations of natriuretic peptides in patients treated percutaneously increased during the first month after ASD closure and normalized thereafter, but in patients treated surgically, they remained higher than in controls. (II): In the PDA group, at baseline, the end-diastolic diameter of LV measured over 2SD in 5 of 33 patients. The median N-terminal pro-brain natriuretic peptide (proBNP) concentration before closure measured 72 ng/l in the control group and 141 ng/l in the PDA group (P = 0.001) and 6 months after closure measured 78.5 ng/l (P = NS). Patients differed from control subjects in indices of LV diastolic and systolic function at baseline, but by the end of follow-up, all these differences had disappeared. Even in the subgroup of patients with normal-sized LV at baseline, the LV end-diastolic volume decreased significantly during follow-up. (III): Before repair, the size and wall thickness of LV were higher in patients with CoA than in controls. Systolic blood pressure measured a median 123 mm Hg in patients before repair (P < 0.001) and 103 mm Hg one year thereafter, and 101 mm Hg in controls. The diameter of the coarctation segment measured a median 3.0 mm at baseline, and 7.9 at the 12-month (P = 0.006) follow-up. Thicknesses of the interventricular septum and posterior wall of the LV decreased after repair but increased to the initial level one year thereafter. The velocity time integrals of mitral inflow increased, but no changes were evident in LV dimensions or contractility. During follow-up, serum levels of natriuretic peptides decreased correlating with diastolic and systolic indices of LV function in 2D and 3D echocardiography. (IV): In 2D echocardiography, the interventricular septum and LV posterior wall were thicker, and velocity time integrals of mitral inflow shorter in patients with Mulibrey nanism than in controls. In 3D echocardiography, LV end-diastolic volume measured a median 51.9 (range 33.3 to 73.4) ml/m² in patients and 59.7 (range 37.6 to 87.6) ml/m² in controls (P = 0.040), and serum levels of ANPN and proBNP a median 0.54 (range 0.04 to 4.7) nmol/l and 289 (range 18 to 9170) ng/l, in patients and 0.28 (range 0.09 to 0.72) nmol/l (P < 0.001) and 54 (range 26 to 139) ng/l (P < 0.001) in controls. They correlated with several indices of diastolic LV function. Conclusions (I): During the one-year follow-up after the ASD closure, RV size decreased but did not normalize in all patients. The size of the LV normalized after ASD closure but the increase in LV size was slower in patients treated surgically than in those treated with the percutaneous technique. Serum levels of ANPN and proBNP were elevated prior to ASD closure but decreased thereafter to control levels in patients treated with the percutaneous technique but not in those treated surgically. (II): Changes in LV volume and function caused by PDA disappeared by 6 months after percutaneous closure. Even the children with normal-sized LV benefited from the procedure. (III): After repair of CoA, the RV size and the velocity time integrals of mitral inflow increased, and serum levels of natriuretic peptides decreased. Patients need close follow-up, despite cessation of LV pressure overload, since LV hypertrophy persisted even in normotensive patients with normal growth of the coarctation segment. (IV): In children with Mulibrey nanism, the LV wall was hypertrophied, with myocardial restriction and impairment of LV function. Significant correlations appeared between indices of LV function, size of the left atrium, and levels of natriuretic peptides, indicating that measurement of serum levels of natriuretic peptides can be used in the clinical follow-up of this patient group despite its dependence on loading conditions.

The role of mast cells in ischemic and hemorrhagic brain injury

Stroke, ischemic or hemorrhagic, belongs among the foremost causes of death and disability worldwide. Massive brain swelling is the leading cause of death in large hemispheric strokes and is only modestly alleviated by available treatment. Thrombolysis with tissue plasminogen activator (TPA) is the only approved therapy in acute ischemic stroke, but fear of TPA-mediated hemorrhage is often a reason for withholding this otherwise beneficial treatment. In addition, recanalization of the occluded artery (spontaneously or with thrombolysis) may cause reperfusion injury by promoting brain edema, hemorrhage, and inflammatory cell infiltration. A dominant event underlying these phenomena seems to be disruption of the blood-brain barrier (BBB). In contrast to ischemic stroke, no widely approved clinical therapy exists for intracerebral hemorrhage (ICH), which is associated with poor outcome mainly due to the mass effect of enlarging hematoma and associated brain swelling. Mast cells (MCs) are perivascularly located resident inflammatory cells which contain potent vasoactive, proteolytic, and fibrinolytic substances in their cytoplasmic granules. Experiments from our laboratory showed MC density and their state of granulation to be altered early following focal transient cerebral ischemia, and degranulating MCs were associated with perivascular edema and hemorrhage. (I) Pharmacological MC stabilization led to significantly reduced ischemic brain swelling (40%) and BBB leakage (50%), whereas pharmacological MC degranulation raised these by 90% and 50%, respectively. Pharmacological MC stabilization also revealed a 40% reduction in neutrophil infiltration. Moreover, genetic MC deficiency was associated with an almost 60% reduction in brain swelling, 50% reduction in BBB leakage, and 50% less neutrophil infiltration, compared with controls. (II) TPA induced MC degranulation in vitro. In vivo experiments with post-ischemic TPA administration demonstrated 70- to 100-fold increases in hemorrhage formation (HF) compared with controls HF. HF was significantly reduced by pharmacological MC stabilization at 3 (95%), 6 (75%), and 24 hours (95%) of follow-up. Genetic MC deficiency again supported the role of MCs, leading to 90% reduction in HF at 6 and 24 hours. Pharmacological MC stabilization and genetic MC deficiency were also associated with significant reduction in brain swelling and in neutrophil infiltration. Importantly, these effects translated into a significantly better neurological outcome and lower mortality after 24 hours. (III) Finally, in ICH experiments, pharmacological MC stabilization resulted in significantly less brain swelling, diminished growth in hematoma volume, better neurological scores, and decreased mortality. Pharmacological MC degranulation produced the opposite effects. Genetic MC deficiency revealed a beneficial effect similar to that found with pharmacological MC stabilization. In sum, the role of MCs in these clinically relevant scenarios is supported by a series of experiments performed both in vitro and in vivo. That not only genetic MC deficiency but also drugs targeting MCs could modulate these parameters (translated into better outcome and decreased mortality), suggests a potential therapeutic approach in a number of highly prevalent cerebral insults in which extensive tissue injury is followed by dangerous brain swelling and inflammatory cell infiltration. Furthermore, these experiments could hint at a novel therapy to improve the safety of thrombolytics, and a potential cellular target for those seeking novel forms of treatment for ICH.

Thrombin regulation in newborn infants

The coagulation system of newborn infants differs markedly from that of older children and adults. The activities of most coagulation factors and anticoagulants are low, leading to altered regulation in the formation of the key enzyme, thrombin. Timely and adequate generation of thrombin is essential, as thrombin activates platelets and many coagulation factors, cleaves fibrinogen into fibrin and activates the antithrombotic and anti-inflammatory protein C pathway. On the other hand, excess thrombin may promote thrombotic complications and exacerbate harmful inflammatory reactions. Despite the characteristic features, the newborn coagulation system can be considered physiological, since healthy newborns rarely show haemorrhagic or thrombotic complications. Sick newborns, however, often encounter clinical situations that challenge their coagulation system. The aim of this study was to clarify the behaviour of the neonatal coagulation system in selected clinical situations, with a special emphasis on the generation of thrombin. Thrombin was measured by in vivo thrombin generation markers and by thrombin generation potential in vitro. The patient groups included sick newborns undergoing intensive care and receiving fresh-frozen plasma (FFP), requiring exchange transfusions (ET) or presenting with a congenital heart defect requiring open heart surgery. Additionally, healthy newborns with inherited heterozygous factor V Leiden (FVL) mutation were studied. Thrombin generation potential was also analysed in cord plasma of healthy infants and in adults. Healthy as well as sick newborn infants showed lower total thrombin generation potential in vitro but faster initiation of thrombin generation than adults. These findings were qualitatively similar when plasma was supplemented with platelets. Platelets, however, significantly altered the effect of the major anticoagulant, activated protein C (APC), on thrombin generation potential. In accordance with previous studies, thrombin generation in healthy newborn platelet-poor plasma was resistant to the anticoagulant effects of APC, but when the plasma was supplemented with platelets APC attenuated thrombin generation significantly more in newborns than in adults. In vivo generation of thrombin was elevated in nearly all of the sick newborn infants. The low-volume FFP transfusion as opposed to the change from neonatal to adult blood in ET exerted markedly different effects on neonatal thrombin generation. FFP reduced the in vivo generation of thrombin in those newborns with the highest pretransfusional thrombin generation, thus acting as an anticoagulant agent. In those infants with lower pretransfusional thrombin generation, the effect of FFP on thrombin generation was fairly neutral. On the other hand, the combination of red blood cells and FFP, used to perform ET, significantly increased the in vivo thrombin formation and shifted the balance in the newborn coagulation system to the procoagulant direction. Cardiopulmonary bypass (CPB) also significantly increased the in vivo thrombin generation, but the thrombin generation profile during CPB differed from that previously observed in adults. Escalation of thrombin at early reperfusion was not observed in newborns; in adults, its occurrence is associated with postoperative myocardial damage. Finally, in healthy newborns with FVL heterozygosity, faster initiation of thrombin generation was observed compared with controls. Interestingly, FV level was lower in FVL-heterozygous infants, possibly to counteract the procoagulant effects induced by FVL. In conclusion, unique features regarding thrombin regulation in newborn infants were observed. These features included a novel platelet effect on the regulation of the protein C pathway. The clinical challenges mainly seemed to shift the balance in the coagulation system of newborns to the procoagulant direction. Blood component transfusions markedly affected coagulation in a manner specific to the product but that could also be altered by the clinical situation. Overall, the results highlight the need for understanding developmental haemostasis for both diagnostic and therapeutic purposes.

Cyclosporine population pharmacokinetics in pediatric renal transplant recipients

Cyclosporine is an immunosuppressant drug with a narrow therapeutic index and large variability in pharmacokinetics. To improve cyclosporine dose individualization in children, we used population pharmacokinetic modeling to study the effects of developmental, clinical, and genetic factors on cyclosporine pharmacokinetics in altogether 176 subjects (age range: 0.36–20.2 years) before and up to 16 years after renal transplantation. Pre-transplantation test doses of cyclosporine were given intravenously (3 mg/kg) and orally (10 mg/kg), on separate occasions, followed by blood sampling for 24 hours (n=175). After transplantation, in a total of 137 patients, cyclosporine concentration was quantified at trough, two hours post-dose, or with dose-interval curves. One-hundred-four of the studied patients were genotyped for 17 putatively functionally significant sequence variations in the ABCB1, SLCO1B1, ABCC2, CYP3A4, CYP3A5, and NR1I2 genes. Pharmacokinetic modeling was performed with the nonlinear mixed effects modeling computer program, NONMEM. A 3-compartment population pharmacokinetic model with first order absorption without lag-time was used to describe the data. The most important covariate affecting systemic clearance and distribution volume was allometrically scaled body weight i.e. body weight**3/4 for clearance and absolute body weight for volume of distribution. The clearance adjusted by absolute body weight declined with age and pre-pubertal children (< 8 years) had an approximately 25% higher clearance/body weight (L/h/kg) than did older children. Adjustment of clearance for allometric body weight removed its relationship to age after the first year of life. This finding is consistent with a gradual reduction in relative liver size towards adult values, and a relatively constant CYP3A content in the liver from about 6–12 months of age to adulthood. The other significant covariates affecting cyclosporine clearance and volume of distribution were hematocrit, plasma cholesterol, and serum creatinine, explaining up to 20%–30% of inter-individual differences before transplantation. After transplantation, their predictive role was smaller, as the variations in hematocrit, plasma cholesterol, and serum creatinine were also smaller. Before transplantation, no clinical or demographic covariates were found to affect oral bioavailability, and no systematic age-related changes in oral bioavailability were observed. After transplantation, older children receiving cyclosporine twice daily as the gelatine capsule microemulsion formulation had an about 1.25–1.3 times higher bioavailability than did the younger children receiving the liquid microemulsion formulation thrice daily. Moreover, cyclosporine oral bioavailability increased over 1.5-fold in the first month after transplantation, returning thereafter gradually to its initial value in 1–1.5 years. The largest cyclosporine doses were administered in the first 3–6 months after transplantation, and thereafter the single doses of cyclosporine were often smaller than 3 mg/kg. Thus, the results suggest that cyclosporine displays dose-dependent, saturable pre-systemic metabolism even at low single doses, whereas complete saturation of CYP3A4 and MDR1 (P-glycoprotein) renders cyclosporine pharmacokinetics dose-linear at higher doses. No significant associations were found between genetic polymorphisms and cyclosporine pharmacokinetics before transplantation in the whole population for which genetic data was available (n=104). However, in children older than eight years (n=22), heterozygous and homozygous carriers of the ABCB1 c.2677T or c.1236T alleles had an about 1.3 times or 1.6 times higher oral bioavailability, respectively, than did non-carriers. After transplantation, none of the ABCB1 SNPs or any other SNPs were found to be associated with cyclosporine clearance or oral bioavailability in the whole population, in the patients older than eight years, or in the patients younger than eight years. In the whole population, in those patients carrying the NR1I2 g.-25385C–g.-24381A–g.-205_-200GAGAAG–g.7635G–g.8055C haplotype, however, the bioavailability of cyclosporine was about one tenth lower, per allele, than in non-carriers. This effect was significant also in a subgroup of patients older than eight years. Furthermore, in patients carrying the NR1I2 g.-25385C–g.-24381A–g.-205_-200GAGAAG–g.7635G–g.8055T haplotype, the bioavailability was almost one fifth higher, per allele, than in non-carriers. It may be possible to improve individualization of cyclosporine dosing in children by accounting for the effects of developmental factors (body weight, liver size), time after transplantation, and cyclosporine dosing frequency/formulation. Further studies are required on the predictive value of genotyping for individualization of cyclosporine dosing in children.

The effect of blood glucose on the vasculature in young patients with type 1 diabetes

Background. Patients with type 1 diabetes are at markedly increased risk of vascular complications. In this respect it is noteworthy that hyperglycaemia that is shown to cause endothelial dysfunction, has clearly been shown to be a risk factor for diabetic microvascular disease. However, the role of hyperglycaemia as a predictor of macrovascular disease is not as clear as for microvascular disease, although type 1 diabetes itself increases the risk of cardiovascular disease substantially. Furthermore, it is not known whether it is the short-term or the long-term hyperglycaemia that confers possible risk. In addition, the role of glucose variability as a predictor of complications is to a large extent unexplored. Interestingly, although hyperglycaemia increases the risk of pre-eclampsia in women with type 1 diabetes, it is unclear whether pre-eclampsia, a condition characterized by endothelial dysfunction, is also a risk factor for microvascular complication, diabetic nephropathy. Aims. This doctoral thesis investigated the role of acute hyperglycaemia and glucose variability on arterial stiffness and cardiac ventricular repolarisation in male patients with type 1 diabetes as well as in healthy male volunteers. The thesis also explored whether acute hyperglycaemia leads to an inflammatory response, endothelial dysfunction and oxidative stress. Finally, the role of pre-eclampsia, as a predictor of diabetic nephropathy in type 1 diabetes was examined. Subjects and methods. In order to study glucose variability and the daily glycaemic control, 22 male patients with type 1 diabetes, without any diabetic complications, were monitored for 72-h with a continuous glucose monitoring system. At the end of the 72-h glucose monitoring period a 2-h hyperglycaemic clamp was performed both in the patients with type 1 diabetes and in the 13 healthy age-matched male volunteers. Blood pressure, arterial stiffness and QT time were measured to detect vascular changes during acute hyperglycaemia. Blood samples were drawn at baseline (normoglycaemia) and during acute hyperglycaemia. In another patient sample, women with type 1 diabetes were followed during their pregnancy and restudied eleven years later to elucidate the role of pre-eclampsia and pregnancy-induced hypertension as potential risk factors for diabetic nephropathy. Results and conclusions. Acute hyperglycaemia increased arterial stiffness as well as caused a disturbance in the myocardial ventricular repolarisation, emphasizing the importance of a strict daily glycaemic control in male patients with type 1 diabetes. An inflammatory response was also observed during acute hyperglycaemia. Furthermore, a high mean daily blood glucose but not glucose variability per se is associated with arterial stiffness. While glucose variability in turn correlated with central blood pressure, the results suggest that the glucose metabolism is closely linked to the haemodynamic changes in male patients with uncomplicated type 1 diabetes. Notably, the results are not directly applicable to females. Finally, a history of a pre-eclamptic pregnancy, but not pregnancy-induced hypertension was associated with increased risk of diabetic nephropathy.

Proton magnetic resonance spectroscopy of a boron neutron capture therapy 10B-carrier, L-p-boronophenylalanine-fructose complex

Boron neutron capture therapy (BNCT) is a radiotherapy that has mainly been used to treat malignant brain tumours, melanomas, and head and neck cancer. In BNCT, the patient receives an intravenous infusion of a 10B-carrier, which accumulates in the tumour area. The tumour is irradiated with epithermal or thermal neutrons, which result in a boron neutron capture reaction that generates heavy particles to damage tumour cells. In Finland, boronophenylalanine fructose (BPA-F) is used as the 10B-carrier. Currently, the drifting of boron from blood to tumour as well as the spatial and temporal accumulation of boron in the brain, are not precisely known. Proton magnetic resonance spectroscopy (1H MRS) could be used for selective BPA-F detection and quantification as aromatic protons of BPA resonate in the spectrum region, which is clear of brain metabolite signals. This study, which included both phantom and in vivo studies, examined the validity of 1H MRS as a tool for BPA detection. In the phantom study, BPA quantification was studied at 1.5 and 3.0 T with single voxel 1H MRS, and at 1.5 T with magnetic resonance imaging (MRSI). The detection limit of BPA was determined in phantom conditions at 1.5 T and 3.0 T using single voxel 1H MRS, and at 1.5 T using MRSI. In phantom conditions, BPA quantification accuracy of ± 5% and ± 15% were achieved with single voxel MRS using external or internal (internal water signal) concentration references, respectively. For MRSI, a quantification accuracy of <5% was obtained using an internal concentration reference (creatine). The detection limits of BPA in phantom conditions for the PRESS sequence were 0.7 (3.0 T) and 1.4 mM (1.5 T) mM with 20 × 20 × 20 mm3 single voxel MRS, and 1.0 mM with acquisition-weighted MRSI (nominal voxel volume 10(RL) × 10(AP) × 7.5(SI) mm3), respectively. In the in vivo study, an MRSI or single voxel MRS or both was performed for ten patients (patients 1-10) on the day of BNCT. Three patients had glioblastoma multiforme (GBM), and five patients had a recurrent or progressing GBM or anaplastic astrocytoma gradus III, and two patients had head and neck cancer. For nine patients (patients 1-9), MRS/MRSI was performed 70-140 min after the second irradiation field, and for one patient (patient 10), the MRSI study began 11 min before the end of the BPA-F infusion and ended 6 min after the end of the infusion. In comparison, single voxel MRS was performed before BNCT, for two patients (patients 3 and 9), and for one patient (patient 9), MRSI was performed one month after treatment. For one patient (patient 10), MRSI was performed four days before infusion. Signals from the tumour spectrum aromatic region were detected on the day of BNCT in three patients, indicating that in favourable cases, it is possible to detect BPA in vivo in the patient’s brain after BNCT treatment or at the end of BPA-F infusion. However, because the shape and position of the detected signals did not exactly match the BPA spectrum detected in the in vitro conditions, assignment of BPA is difficult. The opportunity to perform MRS immediately after the end of BPA-F infusion for more patients is necessary to evaluate the suitability of 1H MRS for BPA detection or quantification for treatment planning purposes. However, it could be possible to use MRSI as criteria in selecting patients for BNCT.

Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.