Bilirubin oxidation products, oxidative stress, and intracerebral hemorrhage.

Hematoma and perihematomal regions after intracerebral hemorrhage (ICH) are biochemically active environments known to undergo potent oxidizing reactions. We report facile production of bilirubin oxidation products (BOXes) via hemoglobin/Fenton reaction under conditions approximating putative in vivo conditions seen following ICH. Using a mixture of human hemoglobin, physiological buffers, unconjugated solubilized bilirubin, and molecular oxygen and/or hydrogen peroxide, we generated BOXes, confirmed by spectral signature consistent with known BOXes mixtures produced by independent chemical synthesis, as well as HPLC-MS of BOX A and BOX B. Kinetics are straightforward and uncomplicated, having initial rates around 0.002 microM bilirubin per microM hemoglobin per second under normal experimental conditions. In hematomas from porcine ICH model, we observed significant production of BOXes, malondialdehyde, and superoxide dismutase, indicating a potent oxidizing environment. BOX concentrations increased from 0.084 +/- 0.01 in fresh blood to 22.24 +/- 4.28 in hematoma at 72h, and were 11.22 +/- 1.90 in adjacent white matter (nmol/g). Similar chemical and analytical results are seen in ICH in vivo, indicating the hematoma is undergoing similar potent oxidations. This is the first report of BOXes production using a well-defined biological reaction and in vivo model of same. Following ICH, amounts of unconjugated bilirubin in hematoma can be substantial, as can levels of iron and hemoglobin. Oxidation of unconjugated bilirubin to yield bioactive molecules, such as BOXes, is an important discovery, expanding the role of bilirubin in pathological processes seen after ICH.

Selective gray matter staining of human brain slices: optimized use of cadaver materials.

We report a novel staining technique for human brain slices that distinguishes clearly gray from white matter. Previously described techniques using either Prussian blue (Berlin blue) or phthalocyanine dyes usually have included a hot phenol pretreatment to prevent white matter staining. The technique we describe here does not require hot phenol pretreatment and allows the use of brains stored for postmortem periods of one to two years prior to staining. Our technique involves staining with copper(II) phthalocyanine-tetrasulfonic acid tetrasodium salt 1% in water for 2 h followed by acetic acid treatment; this produces excellent blue staining of gray matter with little white matter staining. The stained brain slices are excellent for teaching human brain anatomy and/or pathology, or for research purposes.

Height, weight, and motor-social development during the first 18 months of life in 126 infants born to 109 mothers with polycystic ovary syndrome who conceived on and continued metformin through pregnancy.

BACKGROUND: We prospectively assessed growth and motor-social development during the first 18 months of life in 126 live births (122 pregnancies) to 109 women with polycystic ovary syndrome (PCOS) who conceived on and continued metformin (1.5-2.55 g/day) through pregnancy. METHODS: The lengths and weights of PCOS neonates were compared with gender-specific Centers for Disease Control and Prevention (CDC) infant data. Gestational diabetes (GD) and pre-eclampsia in women with PCOS were compared with 252 healthy women without PCOS who had >or=1 live birth (262 live births). RESULTS: There were 101 out of 126 (80%) term (>or=37 gestational weeks) PCOS births, which was not significantly different (P = 0.7) from controls, 206 out of 252 (81.7%). There were two (1.6%) birth defects. GD occurred in nine out of 119 PCOS pregnancies (7.6%) versus 40 out of 251 (15.9%) controls, P = 0.027. The prevalence of pre-eclampsia did not differ in PCOS versus control pregnancies (4.1 versus 3.6%, P = 0.8). The birth length and weight of the 52 male neonates did not differ (P > 0.05) from those of CDC males; the 74 female neonates were shorter than CDC females (48.9 +/- 5.4 versus 50.6 +/- 2.7 cm, P = 0.006) and weighed less (3.09 +/- 0.85 versus 3.29 +/- 0.52 kg, P = 0.04). There were no systematic differences in growth between PCOS and CDC infants over 18 months. At 3, 6, 9, 12 and 18 months, of a potential 100% motor-social development score, scores (+/-SD) were 95 +/- 13, 98 +/- 8%, 95 +/- 10, 97 +/- 8 and 94 +/- 16%; no infants had motor-social developmental delays. CONCLUSIONS: Metformin reduced development of GD, was not teratogenic and did not adversely affect birth length and weight, growth or motor-social development in the first 18 months of life.

Metformin during pregnancy reduces insulin, insulin resistance, insulin secretion, weight, testosterone and development of gestational diabetes: prospective longitudinal assessment of women with polycystic ovary syndrome from preconception throughout pregnancy.

BACKGROUND: In a prospective observational study of 42 pregnancies in 39 Caucasian women (age 30 +/- 4 years) with polycystic ovary syndrome (PCOS), we examined effects of metformin on maternal insulin, insulin resistance (IR), insulin secretion (IS), weight gain, development of gestational diabetes (GD), testosterone and plasminogen activator inhibitor activity. We assessed the hypothesis that diet-metformin (MET) lessens the physiological gestational increase in IR and reduces gestational weight gain, thus reducing GD. METHODS: Preconception, in an out-patient clinical research centre, MET 1.5 (eight pregnancies) to 2.55 g/day (34 pregnancies) was started. Women with body mass index <25 or >or=25 kg/m(2) were given a 2000 or 1500 calorie/day, high-protein (26% of calories), low-carbohydrate (44%) diet. Calorie restrictions were dropped after conception. RESULTS: On MET, GD developed in three out of 42 pregnancies (7.1%). Median entry weight (94.5 kg) fell to 82.7 on MET at the last preconception visit (P = 0.0001), fell further to 81.6 during the first trimester, was 83.6 in the second trimester, and 89.1 kg in the third trimester. Median weight gain during pregnancy was 3.5 kg. The median percentage reduction in serum insulin was 40% on MET at the last preconception visit; insulin did not increase in the first or second trimesters (P > 0.05), and rose 10% in the third trimester. The median percentage reduction in HOMA IR was 46% on MET at the last preconception visit; IR did not increase (P > 0.05) in the first, second or third trimesters. HOMA insulin secretion fell 45% on MET at the last preconception visit, did not increase in the first trimester, rose 24% in the second trimester, and rose 109% in the third trimester. Testosterone fell 30% on MET at the last preconception visit (P = 0.01) and then rose 74, 61 and 95% during trimesters 1, 2 and 3; median testosterone during the third trimester did not differ from pre-treatment levels. CONCLUSIONS: By reducing preconception weight, insulin, IR, insulin secretion and testosterone, and by maintaining these insulin-sensitizing effects throughout pregnancy, MET-diet reduces the likelihood of developing GD, and prevents androgen excess for the fetus.