Ursula Turpeinen

Evolution of Cerebral Tumor Necrosis Factor-α Production During Human Ischemic Stroke

Background and Purpose— Tumor necrosis factor-&agr; (TNF-&agr;) is detected in ischemic brain cells in experimental animal models and is believed to play an important role in apoptosis. However, the natural expression of TNF-&agr; during human stroke is not known. Methods— We examined TNF-&agr; immunohistochemistry and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) in brain samples of stroke victims (n=16) after variable survival (15 hours to 18 days). Systemic TNF-&agr; content from a separate cohort including severe or lethal stroke cases (n=26) was also assayed. Results— Neuronal TNF-&agr; was demonstrated from 0.6 to 5.4 days after the onset of stroke symptoms, peaking bilaterally during days 2 and 3. Bilateral glial TNF-&agr; immunoreactivity was detected during the acute phase, with the astrocytic TNF-&agr; expression dominating in later phases and persisting contralaterally to the infarct in more matured phases (17 to 18 days). Invading inflammatory cells were TNF-&agr; immunopositive beginning on the third day. Besides, vascular wall structures showed immunoreactivity sporadically. TNF-&agr; levels were mostly nondetectable in peripheral blood. TUNEL labeling and TNF-&agr; staining overlapped, although not completely, during the first days. Conclusions— The data support the hypothesis that TNF-&agr; may be involved both in the acute propagation of inflammatory processes and cell death and possibly in the more delayed reconstitutive processes of human ischemic stroke.

Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay

Vitamin D status is usually assessed by measuring the serum concentration of 25-hydroxyvitamin D [25(OH)D]. Its measurement is important as a clinical indicator of nutritional vitamin D deficiency, which is one of the causes of osteoporosis (1). Vitamin D exists in two forms: cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). Vitamin D2 is further metabolized to 25(OH)D2. Vitamin D3 is formed in the skin from its precursor 7-dehydrocholesterol after ultraviolet irradiation or is absorbed from the diet (2). It is further hydroxylated in the liver to 25(OH)D3 as the first step of its conversion in the kidney to 1,25-dihydroxyvitamin D3, which is the biologically active form. 25(OH)D3 is the main circulating form of vitamin D. Clinically it is important to measure both forms of 25-hydroxyvitamin D to monitor the effect of vitamin D2 supplementation on total vitamin D status. The first routine methods for measurement of 25(OH)D concentrations in human plasma were based on competitive protein binding and used vitamin D-binding protein and a tritium-labeled tracer (3). These methods were replaced by a simpler, rapid RIA (4), and a radioiodinated tracer was incorporated into the RIA in 1993 (5). This assay principle is the basis of several commercially available methods. Quantitative HPLC assays have been developed based on ultraviolet detection and normal-phase separation (6), combined use of normal- and reversed-phase separations (7), or reversed-phase separation alone(8). Recently, reversed-phase HPLC methods for 25(OH)D3 in human plasma have been developed with normal-phase prepurification of the sample (9) or liquid extraction only (10). Earlier HPLC methods for 25(OH)D3 in serum were designed mainly for research purposes and were therefore too complicated for routine use. The present method was designed to be easy to use, sensitive, and rapid with …

Postmenopausal Hormone Replacement Therapy Modifies Skeletal Muscle Composition and Function: A Study with Monozygotic Twin Pairs

We investigated whether long-term hormone replacement therapy (HRT) is associated with mobility and lower limb muscle performance and composition in postmenopausal women. Fifteen 54- to 62-yr-old monozygotic female twin pairs discordant for HRT were recruited from the Finnish Twin Cohort. Habitual (HWS) and maximal (MWS) walking speeds over 10 m, thigh muscle composition, lower body muscle power assessed as vertical jumping height, and maximal isometric hand grip and knee extension strengths were measured. Intrapair differences (IPD%) with 95% confidence intervals (CI) were calculated. The mean duration of HRT use was 6.9 +/- 4.1 yr. MWS was on average 7% (0.9 to 13.1%, P = 0.019) and muscle power 16% (-0.8 to 32.8%, P = 0.023) greater in HRT users than in their cotwins. Thigh muscle cross-sectional area tended to be larger (IPD% = 6%, 95% CI: -0.07 to 12.1%, P = 0.065), relative muscle area greater (IPD% = 8%, CI: 0.8 to 15.0%, P = 0.047), and relative fat area smaller (IPD% = -5%, CI: -11.3 to 1.2%, P = 0.047) in HRT users than in their sisters. There were no significant differences in maximal isometric strengths or HWS between users and nonusers. Subgroup analyses revealed that estrogen-containing therapies (11 pairs) significantly decreased total body and thigh fat content, whereas tibolone (4 pairs) tended to increase muscle cross-sectional area. This study showed that long-term HRT was associated with better mobility, greater muscle power, and favorable body and muscle composition among 54- to 62-yr-old women. The results indicate that HRT is a potential agent in preventing muscle weakness and mobility limitation in older women.

Increased Activity of the Hypothalamic-Pituitary-Testicular Axis in Infancy Results in Increased Androgen Action in Premature Boys

CONTEXT Transient activation of the hypothalamic-pituitary-gonadal (HPG) axis is observed in boys during the first months of life. Previous research suggests increased HPG axis activation in premature infants, but the physiological significance of this has not been studied. OBJECTIVE The objective of this study was to evaluate the differences in reproductive hormone levels and their biological effects between full-term (FT) and preterm (PT) infant boys. STUDY DESIGN AND PARTICIPANTS Twenty-five FT and 25 PT (gestational age 24.7-36.6 wk) boys were recruited at birth and followed up monthly from 1 wk to 6 months of age (d 7, months 1-6). Nineteen FT and 20 PT boys were reexamined at 14 months of age. MAIN OUTCOME MEASURES Urinary gonadotropins and testosterone were measured in serial urine samples and compared with testicular and penile growth. Urinary prostate-specific antigen was measured as an androgen biomarker. RESULTS LH and testosterone levels were higher in PT boys (P < 0.001 for both) than FT boys. Compared with FT boys, FSH levels were lower at d 7 (P = 0.002) but higher from month 1 to month 3 (P = 0.002-0.030) in PT boys. This was associated with significantly faster testicular and penile growth in PT boys compared with FT boys. Transient increase in the prostate-specific antigen levels in both groups indicated androgen action in the prostate. CONCLUSIONS Postnatal HPG axis activation in infancy is increased in PT boys and associated with faster testicular and penile growth compared with FT boys. Possible long-term consequences of hyperandrogenism in PT infant boys warrant further research.

Automated Measurement of 25-OH Vitamin D3 on the Roche Modular E170 Analyzer

BACKGROUND The first commercial direct automated immunoassay specific for 25-OH vitamin D(3) (25-OH-D(3)) was recently introduced for use on Roche Diagnostics immunoassay analyzers. We assessed the analytical performance of the Elecsys 25-OH-D(3) assay on a Roche Modular E 170 analyzer. METHODS The Elecsys 25-OH-D(3) assay is a direct electrochemiluminescence immunoassay for human serum or plasma. It is a competitive assay in which the binding protein of vitamin D is inactivated during incubation. The assay employs a polyclonal antibody directed against 25-OH vitamin D(3). We compared the 25-OH-D(3) assay to assays performed with RIA, HPLC, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS At concentrations of 48, 76, and 124 nmol/L, within-run CVs were 5.1%, 3.1%, and 7.1% and total CVs were 12.1%, 7.4%, and 10.6%, respectively. A comparison of Elecsys 25-OH vitamin D(3) with RIA yielded the regression equation: Elecsys = 1.114 x RIA - 6.15 (S(y|x) = 15.7 nmol/L; n = 163). The corresponding equation with HPLC was: Elecsys = 1.077 x HPLC + 5.442 (S(y|x) = 13.9 nmol/L; n = 67) and with LC-MS/MS: Elecsys = 0.887 x LC-MS/MS + 5.046 (S(y|x) =12.4 nmol/L; n = 64). Contrary to LC-MS/MS, with the cutoff of 50 nmol/L (deficiency vs normal), approximately 10% of samples were misclassified as normal with RIA and Elecsys. Plasma samples were observed to have markedly higher concentrations than serum samples. CONCLUSIONS The Elecsys concentrations of 25-OH-D(3) were in good overall agreement with those determined with LC-MS/MS and RIA. However, large between-method variation was observed in individual patient samples. Use of serum rather than plasma is preferred owing to the higher results observed with plasma samples.

Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry.

Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable‐isotope dilution liquid chromatography‐tandem mass spectrometric (LC‐MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether‐ethyl acetate from 250 µL or 500 µL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple‐reaction monitoring (MRM) mode after separation on a reversed‐phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d2 testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2–100 nmol/L. Interassay CVs were 4.2–7.6 % at mean concentrations of testosterone of 3.3–45 nmol/L. Total measurement uncertainty (U, k = 2) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal‐to‐noise ratio = 3) and the overall method recovery of testosterone was 95 %. Correlation (r) with our in‐house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18–30, 31–50, 51–70 and over 70 years were established. Sensitivity and specificity of the LC‐MS/MS method offer advantages over immunoassay and make it suitable for use as a high‐throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.

Failure of the combination of sequential oral and transdermal estradiol plus norethisterone acetate to affect plasma homocysteine levels

OBJECTIVE A high level of plasma homocysteine may be deleterious to vascular health. We therefore compared the effect of combinations of sequential oral and transdermal estradiol plus norethisterone acetate on plasma homocysteine. DESIGN Prospective, randomized study. SETTING Outpatient department of obstetrics and gynecology in a university hospital. PATIENT(S) Forty-two healthy, nonsmoking postmenopausal women starting hormone replacement therapy (HRT) to control climacteric symptoms. INTERVENTION(S) In a randomized order, the women started using either oral HRT (2 mg of estradiol on days 1-12, 2 mg of estradiol plus 1 mg of norethisterone acetate (NETA) on days 13-22, and 1mg of estradiol on days 23-28; n = 21) or transdermal HRT (50 microg/d of estradiol on days 1-28 and 250 microg/d of norethisterone acetate on days 15-28, n = 21) for 1 year. MAIN OUTCOME MEASURE(S) Fasting plasma levels of homocysteine were measured before the treatment and during the combined estradiol-plus-NETA phases of the sixth and 12th treatment cycles. RESULT(S) Basal homocysteine levels in the oral group (8.2 +/- 3.1 micromol/L, mean plusmn;SD) and transdermal group (8.7 plusmn; 1.8 micromol/L, mean plusmn;SD) were not affected by the estradiol-plus-NETA combination. CONCLUSION(S) Neither an oral nor a transdermal combination of sequential estradiol and NETA causes significant changes in plasma homocysteine in Finnish postmenopausal women with normal baseline homocysteine levels.

Cardiac Hypertrophy and Altered Hemodynamic Adaptation in Growth-Restricted Preterm Infants

The objective was to elucidate hemodynamic adaptation in very low birth weight (<1500 g) infants after intrauterine growth retardation. 31 growth-retarded (SGA, birth weight <-2 SD) and 32 appropriate for gestational age (AGA, birth weight within ± 1 SD range) infants were enrolled. In SGA infants, the diastolic diameters of the interventricular septum and the left ventricle were increased, and serum brain natriuretic peptide (BNP) was elevated. Left ventricular output (LVO) of the AGA infants increased from 150 ± 28 to 283 ± 82 mL/kg/min during the study (p < 0.01). The SGA infants had a higher initial LVO than the AGA infants (243 ± 47 versus 150 ± 28 mL/kg/min, p < 0.05), but did not show further LVO increase during the study period. Red cell (RCV) and blood (BV) volume were assessed by Hb subtype analysis, when packed donor red cells were transfused. RCV and BV did not differ between the groups initially, but RCV increased by 18% and BV by 29% in the AGA group during the first 3 d. On day 3, AGA infants had larger BV than the SGA infants (88 ± 5 versus 73 ± 12 mL/kg, p < 0.05). In conclusion, cardiac hypertrophy, elevated initial LVO and BNP of the SGA infants suggest increased cardiac workload after intrauterine growth retardation. Based on the BV and RCV data, blood volume regulation may also be impaired. The data suggest that SGA preterm infants may be exposed to an increased risk of circulatory failure during early adaptation.

Low levels of insulin‐like growth factor‐I in cerebrospinal fluid in children with autism

Autism is a behaviourally defined syndrome characterized by disturbances of social interaction and communication and restrictions of behaviour patterns and imagination. The pathogenesis of autism is unknown but it is suspected that a number of genetic factors may be involved. Neurotrophic factors such as insulin-like growth factor-I (IGF-I) play a role in early brain development. The aim of this study was to determine whether IGF-I levels might be associated with the development of autism. IGF-I levels were measured in the CSF of 11 children with autism (4 females, 7 males; mean age 3.8 years, SD 1.1) using a sensitive radioimmunoassay method and compared with levels in 11 control participants (6 females, 5 males; mean age 3.8 years). Levels of IGF-I in the CSF were statistically significantly lower in the children with autism than in the control children (p=0.03). IGF-I may play a role in pathogenetic mechanisms of autism and the role of neurotrophic factors in autism and other neurodevelopmental diseases should be studied further.

Cerebrospinal fluid insulin-like growth factors IGF-1 and IGF-2 in infantile autism

There has been little exploration of major biologic regulators of cerebral development in autism. We measured insulin-like growth factors (IGF) -1 and -2 from cerebrospinal fluid (CSF) by radio immunoassay in 25 children with autism (median age 5y 5mo; range 1y 11mo-15y 10mo; 20 males, 5 females), and in 16 age-matched comparison children without disability (median age 7y 4mo; range 1y 1mo-15y 2mo; eight males, eight females). IGF-1 and -2 concentrations were further correlated with age of patients and head size. CSF IGF-1 concentration was significantly lower in patients with autism than in the comparison group. The CSF concentrations of children with autism under 5 years of age were significantly lower than their age-matched comparisons. The head circumferences correlated with CSF IGF-1 in children with autism but no such correlation was found in the comparison group. There was no difference between the two groups in CSF IGF-2 concentrations. No patients with autism had macrocephaly. We conclude that low concentrations of CSF IGF-1 at an early age might be linked with the pathogenesis in autism because IGF-1 is important for the survival of Purkinje cells of the cerebellum. The head growth might be explained by the actions of IGF-1 and -2 reflected in CSF concentrations.