Prevalence of Polycystic Ovary Syndrome Phenotypes Using Updated Criteria for Polycystic Ovarian Morphology: An Assessment of Over 100 Consecutive Women Self-reporting Features of Polycystic Ovary Syndrome.

The prevalence of polycystic ovary syndrome (PCOS) and its distinct clinical phenotypes were assessed using 3 sets of international diagnostic criteria in women self-reporting concerns over outward features of PCOS. Revised ultrasonographic criteria for polycystic ovaries (PCO) based on modern ultrasound technology were used. Of the participants, 53%, 62%, and 70% were diagnosed with PCOS using National Institutes of Health, Androgen Excess and PCOS Society, and Rotterdam criteria, respectively. Prevalence of Frank, Ovulatory, Normoandrogenic, and Non-PCO PCOS were 66%, 13%, 11%, and 9%, respectively. Frank PCOS was associated with the severest metabolic disturbances whereas metabolic profiles in Normoandrogenic PCOS did not differ from controls, supporting reduced health risks in women without androgen excess. Metabolic disturbances and hyperandrogenism were linked to excess adiposity across all the groups. Using updated criteria for PCO, the prevalence of Non-PCO PCOS and PCO alone in healthy women recruited from the general population was reduced compared to the previous reports.

Vitamin D levels and relative insufficiency in Saskatchewan.

OBJECTIVE: To determine the extent of vitamin D3 deficiency and levels in pregnant First Nations and non-First Nations women in SK. Also, to determine the distribution of vitamin D3 values in the general population in SK. METHODS: Vitamin D3 levels were measured by LC-MS/MS from 19,181 consecutive patient blood/serum samples received at the Saskatchewan Disease Control Laboratory, and from 743 First Nations, and 301 non-First Nations pregnant women in SK. RESULTS: The ages of the 19,181 patient samples ranged from day 1 (0 years) to 102 years. Of the total, 14,658 were female, and 4523 were males. 30.8% had relative vitamin D3 insufficiency (50-75 nmol/L), and 22.5% were in the deficient range (<50 nmol/L). In summer, a larger percentage of SK patients are in the optimum range, whereas in winter, the number of patients in the vitamin D3 deficiency range increased to 33.0% from 14.1%. Samples from pregnant women were collected during the first trimester of pregnancy. Whereas non-First Nations pregnant women had similar vitamin D3 levels to non-pregnant women in SK, vitamin D3 levels were significantly lower than the optimum of 75 nmol/L in pregnant First Nations women than in non-First Nations women. 29.7% of First Nations pregnant women were in the relative insufficiency range, and 45.6% were vitamin D3 deficient. CONCLUSIONS: First Nations pregnant women have lower vitamin D3 levels than non-First Nations pregnant women. This puts them and their unborn babies at high risk of a diverse range of disorders associated with vitamin D3 deficiency or insufficiency.

LC-MS/MS techniques for high-volume screening of drugs of abuse and target drug quantitation in urine/blood matrices.

Liquid chromatography-tandem mass spectrometry, employing electrospray ionization (ESI), has been applied in the analysis of many drugs and drug metabolites. Sample preparation has been an important part of this technique when analyzing biological samples. Here we describe a high-volume urine screening technique for approximately 40 different drugs of abuse as well as methods for quantification of many other drugs in serum, plasma, and whole blood. These techniques can be used in many different settings from clinical and forensic toxicology examinations to pharmacokinetic studies. Sample preparation procedures range from simple "dilute and shoot" methods to more extensive solid-phase extraction techniques.

Opiate screening and quantitation in urine/blood matrices using LC-MS/MS techniques.

Here we describe a high-volume urinary screening technique for opiate drugs as well as other narcotic analgesics. We also describe methods for quantification of the same drug species in serum, plasma, and whole blood. Screening and quantitation of these types of drugs have presented many challenges, among them the potentially low levels in both abuse and therapeutic situations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing electrospray ionization (ESI), has been able to provide the sensitivity needed for the analysis of many drugs and metabolites. These techniques can be used in many different settings from clinical and forensic toxicology examinations to pharmacokinetic studies and, with appropriate considerations, be applied to different sample matrices. Sample preparation procedures range from simple "dilute and shoot" methods to more extensive solid-phase extraction techniques.

Folic acid transport to the human fetus is decreased in pregnancies with chronic alcohol exposure.

BACKGROUND: During pregnancy, the demand for folic acid increases since the fetus requires this nutrient for its rapid growth and cell proliferation. The placenta concentrates folic acid into the fetal circulation; as a result the fetal levels are 2 to 4 times higher than the maternal level. Animal and in vitro studies have suggested that alcohol may impair transport of folic acid across the placenta by decreasing expression of transport proteins. We aim to determine if folate transfer to the fetus is altered in human pregnancies with chronic alcohol consumption. METHODOLOGY/PRINCIPAL FINDINGS: Serum folate was measured in maternal blood and umbilical cord blood at the time of delivery in pregnancies with chronic and heavy alcohol exposure (n = 23) and in non-drinking controls (n = 24). In the alcohol-exposed pairs, the fetal:maternal serum folate ratio was ≤ 1.0 in over half (n = 14), whereas all but one of the controls were >1.0. Mean folate in cord samples was lower in the alcohol-exposed group than in the controls (33.15 ± 19.89 vs 45.91 ± 20.73, p = 0.04). CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that chronic and heavy alcohol use in pregnancy impairs folate transport to the fetus. Altered folate concentrations within the placenta and in the fetus may in part contribute to the deficits observed in the fetal alcohol spectrum disorders.

A polymerase chain reaction-based genotyping assay for detecting a novel Sandhoff disease-causing mutation.

Sandhoff disease is a rare genetic disorder, however, some northern Saskatchewan communities have a high incidence of the disease (for which the causative mutation has not been described). We discovered a novel mutation causing Sandhoff disease in this community and validated a molecular assay to detect the mutant allele. DNA sequencing was used to search for mutations in the HEXB gene from the most recently affected patient. A polymerase chain reaction (PCR)-based genotyping assay was subsequently designed and validated to detect a novel single-nucleotide deletion using DNA isolated from newborn screening cards. The c.115delG mutation was found in exon 1 of the HEXB gene from 4 patients with clinical presentation of Sandhoff disease. Herein we describe a novel HEXB mutation that is shared among 4 patients with Sandhoff disease, as well as a validated PCR-based genotyping assay that can reliably detect the mutant allele. Because the 4 patients from this community share a common c.115delG mutation in the coding region of the HEXB gene, it may be possible to offer an effective preventive screening program for Sandhoff disease using this assay.

Digit ratios by computer-assisted analysis confirm lack of anatomical evidence of prenatal androgen exposure in clinical phenotypes of polycystic ovary syndrome.

BACKGROUND: We recently showed that women with four clinical phenotypes of polycystic ovary syndrome (PCOS) do not demonstrate anatomical evidence of elevated prenatal androgen exposure as judged by a lower ratio of the index (2D) to ring (4D) finger. However, those findings conflicted with a previous study where women with PCOS had lower right hand 2D:4D compared to healthy female controls. Both these studies used Vernier calipers to measure finger lengths--a method recently shown to be less reliable at obtaining finger length measurements than computer-assisted analysis. METHODS: Ninety-six women diagnosed with PCOS according to the 2003 Rotterdam criteria had their finger lengths measured with computer-assisted analysis. Participants were categorized into four recognized phenotypes of PCOS and their 2D:4D compared to healthy female controls (n = 48) and men (n = 50). RESULTS: Digit ratios assessed by computer-assisted analysis in women with PCOS did not differ from female controls, but were significantly lower in men. When subjects were stratified by PCOS phenotype, 2D:4D did not differ among phenotypes or when compared to female controls. CONCLUSION: Computer-assisted measurements validated that digit ratios of women with PCOS do not show anatomical evidence of increased prenatal androgen exposure.

Elevated neonatal 3-OH isovalerylcarnitine due to breast milk sources in maternal 3-MCC deficiency.

We report a positive newborn screen for 3-hydroxyisovalerylcarnitine (C(5)OH) with an absence of 3-methylcrotonyl-coenzyme A carboxylase deficiency in the neonate. Subsequent blood tests demonstrated persistently elevated C(5)OH. Serial testing of the mother identified markedly elevated C(5)OH in both maternal blood and breast milk. High C(5)OH milk concentrations provide a significant source of C(5)OH to the nursing neonate and possibly explains its persistent elevation in the neonate, a commonly observed finding in maternal 3-MCC deficiency.

Diagnosis and high incidence of hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome in northern Saskatchewan.

Mutations in the SLC25A15 gene, encoding the human inner mitochondrial membrane ornithine transporter, are thought to be responsible for hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome, a rare autosomal recessive condition. HHH syndrome has been detected in several small, isolated communities in northern Saskatchewan (SK). To determine the incidence of HHH syndrome in these communities, a PCR method was set up to detect F188Δ, the common French-Canadian mutation. Neonatal blood spots collected from all newborns from the high risk area were genotyped for the F188Δ mutation for seven consecutive years. Using DNA analysis, we estimated that the heterozygote frequency for the mutant allele for HHH syndrome to be about 1 in 19 individuals, predicting one affected child with HHH syndrome for approximately every 1,500 individuals (1 in 1,550 live births; 1 child every 12 years) in this isolated population. The frequency for the mutant allele for HHH syndrome in this isolated community is probably the highest in the world for this rare disorder. We determined that ornithine levels, by tandem mass spectrometry, were not abnormal in newborns with F188Δ mutation, carriers and normals. Ornithine rises to abnormally high levels at some time after birth well past the time that the newborn screening blood spot is collected. The timing or the reasons for the delayed rise of ornithine in affected children with HHH syndrome have not been determined. Newborn screening for HHH Syndrome in this high risk population is only possible by detection of the mutant allele using DNA analysis.

Digit ratios do not serve as anatomical evidence of prenatal androgen exposure in clinical phenotypes of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is heterogeneous in its clinical presentation and four major phenotypes have been identified. The precise etiology of PCOS is unknown; however, variable exposure to prenatal androgens may be responsible for the spectrum of endocrine and metabolic disturbances characteristic of this syndrome. Since prenatal testosterone exposure is known to decrease the ratio of the second to fourth finger lengths (2D:4D), we characterized the left and right hand 2D:4D in women with clinical variants of PCOS. We hypothesized that if prenatal androgens were involved in the development of the phenotypic spectrum of PCOS, then lower 2D:4D would be differentially expressed among clinical variants of the syndrome. METHODS: Digit ratios were determined in 98 women diagnosed with PCOS by the 2003 international consensus guidelines and in 51 women with regular menstrual cycles, no clinical or biochemical signs of hyperandrogenism and normal ovarian morphology. Women with PCOS were categorized into four clinical phenotypes (i.e. Frank, Non-PCO, Ovulatory and Mild) and 2D:4D among groups were compared by Tukey-Kramer multiple comparisons tests. RESULTS: Left (P = 0.77) and right (P = 0.68) hand 2D:4D were similar among the four clinical phenotypes and no phenotype of PCOS demonstrated a 2D:4D that differed from controls (Left Hand, P = 0.44 and Right Hand, P = 0.75). CONCLUSIONS: Women with PCOS do not demonstrate finger length patterns that are consistent with increased prenatal androgen exposure. These findings do not preclude a role for prenatal androgens in the development of PCOS; however, low 2D:4D are not a characteristic of PCOS.

Drugs of abuse testing by tandem mass spectrometry: a rapid, simple method to replace immunoassays.

PRIMARY OBJECTIVE: To replace immunoassay screening for drugs of abuse (DOA) with a cost-effective tandem mass spectrometry method. SECONDARY OBJECTIVE: To substantially expand the drugs of abuse assay menu. DESIGN AND METHODS: The requirement was to perform high throughput DOA screening for 200 urine specimens/day for 40 drugs/metabolites. The total analysis time had to be <5 min. We used UPLC chromatography, small particle size LC columns and fast scanning tandem mass spectrometry. Urine samples were hydrolyzed enzymatically, diluted and injected with isotopically labeled internal standards. The data produced was transferred by exporting reports as text files to a LIMS system followed by auto certification of the results. RESULTS: 40 different drugs were separated by UPLC (ultra pressure liquid chromatography) with a run time of 5.2 min. Detection limits were below our cut-off values. Individual drug species instead of drug classes were identified; correlation with GC/MS was excellent. A high throughput, robust assay with acceptable accuracy, precision and specificity was developed. The procedure can also be used as a quantitative method with simple modifications. CONCLUSIONS: An improved, high throughput, cost-effective method for drugs of abuse screening has been implemented. GC/MS confirmations were reduced or eliminated. The new procedure is a viable alternative to our previous immunoassay method. Acceptable turn around times, an expanded menu, simplified sample preparation and analytical reliability makes this method a desirable option in the clinical laboratory setting.

Formate pharmacokinetics during formate administration in folate-deficient young swine.

The objective of the study was to investigate the effect of folate deficiency on formate pharmacokinetics during formate administration in folate-deficient young swine. Methanol is a one of the congeners found in alcoholic beverages. Methanol toxicity is mediated through formic acid and thus plays a significant role in the pathophysiology of alcoholism. Folate is a required cofactor in the metabolism of formate to CO(2) and H(2)O. We investigate the effect of folate deficiency on the pharmacokinetics of formate. Twelve young pigs were pair-matched and randomly placed into 2 groups on acquisition ( approximately 5 weeks). One group was made folate deficient (FFD) by feeding with a folic acid-deficient diet; the other group (FFC) was fed a diet supplemented with folate. Four animals (31-38 kg) from each group were infused (intravenous) with 351 mg/kg of sodium formate. The remaining 2 animals were infused with isotonic sodium chloride solution. Blood samples were collected before and at 10, 20, 30, 45, 60, 90, 120, 180, 240, and 480 minutes post dose and analyzed for formate levels by gas chromatography. Pharmacokinetic parameters were estimated using a noncompartmental approach. Formate (mean +/- SE) accumulation was higher in the FFD group than the FFC group (AUC(0-infinity) of 72.37 +/- 8.29 vs 30.08 +/- 2.58 g min/L, respectively). Elimination was also slower in the FFD group (FFD systemic clearance = 0.12 +/- 0.01 L/min compared with FFC systemic clearance = 0.27 +/- 0.02 L/min). Half-life of elimination was 2.5 times longer in FFD group (113 +/- 1 minute) than in FFC group (45 +/- 6 minutes). Folate deficiency had no influence on the volume of distribution of formate (18.84 +/- 1.05 L in FFD vs 17.21 +/- 1.35 L in FFC). Adequate folate status is important in the elimination of formate. A folate-deficiency state results in a reduction in formate elimination kinetics, which may increase the risk of formate toxicity.

Mass spectrometric and physiological validation of a sensitive, automated, direct immunoassay for serum estradiol using the Architect.

BACKGROUND: Measurement of estradiol (E(2)) plays a critical role in the diagnosis and clinical management of reproductive disorders. The challenge for all currently available direct methods for measuring E(2) is to provide accuracy and precision across a wide dynamic range. METHODS: We describe the development and multi-site performance evaluation of a direct E(2) assay on the Architect i2000. Assay performance and method comparisons were performed by testing specimens from men, healthy women with regular menstrual cycles, and post-menopausal women using the Architect assay and isotope dilution, gas chromatography-mass spectrometry (ID/GC-MS). Reference intervals were established by testing prospectively collected daily blood draws from 42 healthy women, 72 postmenopausal women and 101 males. RESULTS: No unexpected cross-reactivity or interference was observed for over 40 compounds tested. Recovery was 100+/-10% in the presence of estrone and estriol. Functional sensitivity (%CV<20%) was <15 pg/ml.(1) The imprecision of the assay was <7.1% (total CV), <2.5%, and <2.3% for control sera containing 45, 190, and 600 pg/ml estradiol, respectively. The assay had a correlation of y=1.033 x+0.3156, r(2)=0.99, n=131 compared to ID/GC-MS. Reference intervals for the current Architect Estradiol assay are reported. CONCLUSIONS: Format changes resulted in dramatic improvement in the performance and accuracy of this direct, fully automated assay. The assay is standardized by ID/GC-MS. The assay is clinically useful for serum concentrations from 15 to >4000 pg/ml.

Formic acid, a novel metabolite of chronic ethanol abuse, causes neurotoxicity, which is prevented by folic acid.

BACKGROUND: Methanol is endogenously formed in the brain and is present as a congener in most alcoholic beverages. Because ethanol is preferentially metabolized over methanol (MeOH) by alcohol dehydrogenase, it is not surprising that MeOH accumulates in the alcohol-abusing population. This suggests that the alcohol-drinking population will have higher levels of MeOH's neurotoxic metabolite, formic acid (FA). FA elimination is mediated by folic acid. Neurotoxicity is a common result of chronic alcoholism. This study shows for the first time that FA, found in chronic alcoholics, is neurotoxic and this toxicity can be mitigated by folic acid administration. OBJECTIVE: To determine if FA levels are higher in the alcohol-drinking population and to assess its neurotoxicity in organotypic hippocampal rat brain slice cultures. METHODS: Serum and CSF FA was measured in samples from both ethanol abusing and control patients, who presented to a hospital emergency department. FA's neurotoxicity and its reversibility by folic acid were assessed using organotypic rat brain hippocampal slice cultures using clinically relevant concentrations. RESULTS: Serum FA levels in the alcoholics (mean +/- SE: 0.416 +/- 0.093 mmol/l, n = 23) were significantly higher than in controls (mean +/- SE: 0.154 +/- 0.009 mmol/l, n = 82) (p < 0.0002). FA was not detected in the controls' CSF (n = 20), whereas it was >0.15 mmol/l in CSF of 3 of the 4 alcoholic cases. Low doses of FA from 1 to 5 mmol/l added for 24, 48 or 72 hours to the rat brain slice cultures caused neuronal death as measured by propidium iodide staining. When folic acid (1 micromol/l) was added with the FA, neuronal death was prevented. CONCLUSIONS: Formic acid may be a significant factor in the neurotoxicity of ethanol abuse. This neurotoxicity can be mitigated by folic acid administration at a clinically relevant dose.

Clinical determination of 17-hydroxyprogesterone in serum by LC-MS/MS: comparison to Coat-A-Count RIA method.

17alpha-Hydroxyprogesterone is a metabolic precursor of cortisol; elevated levels of 17alpha-hydroxyprogesterone are indicative of congenital adrenal hyperplasia. Traditional determination by immunoassay is plagued by poor antibody specificity, resulting in significant interferences. This study explores an LC-MS/MS method for the quantitation of 17OHP in serum. Deuterated 17alpha-hydroxyprogesterone was added as internal standard, followed by solid-phase extraction, HPLC separation with a C16-amide reverse-phase column with run time of 7 min, and quantification by MS/MS (positive electrospray ionisation) in the selected reaction monitoring mode (SRM). Transitions monitored were 331>109 for the analyte and 339>113 for the deuterated internal standard. Intra-assay precision (%R.S.D.) was 7.4% at 7 nmol/L, inter-assay precision (%R.S.D.) at 2, 7 and 27 nmol/L was 15.4, 10.0 and 7.9% and accuracy at 0.9 nmol/L was 100%. The method was linear from 0.156 to 80 nmol/L. Lower limit of quantitation was 0.2 nmol/L, providing meaningful data for patients within normal range as well as those with elevated levels.

Assay of neonatal TSH from blood spots using the Architect i2000, an immunoassay analyzer.

OBJECTIVES: To assess the Architect i2000 for measuring TSH from neonatal blood spots. METHODS: Blood collected on filter paper was eluted and assayed on the Architect. RESULTS: Recovery of TSH was close to 100%. Within-run and between-run precisions were 4.2-8.2% and 14.5-18.1%. Correlation with the Wallac Delphia method was excellent (r = 0.96). Linearity, sensitivity, and slope were 0-400 mU/l, 0.15 mU/l, and 0.86, respectively. The results obtained by this method on the Architect allowed us to differentiate normals from affected patients with the same reliability as the previous method. CONCLUSIONS: Measurement of TSH on the Architect is an excellent method for detecting congenital hypothyroidism.

Lipid peroxidation and protein modification in a mouse model of chronic iron overload.

Iron-storage diseases are believed to cause organ damage through generation of reactive oxygen species. Using a murine model of iron overload, we found that hepatic iron stores increased logarithmically during 3 weeks of chronic intraperitoneal administration of iron dextran, while hepatic glutathione peroxidase activity declined linearly by approximately 50% during the same period. Plasma concentrations of aliphatic aldehydes increased by 2- to 3-fold, and plasma malondialdehyde (MDA) by 6-fold. Modification of total liver protein by products of lipid peroxidation, including MDA-lysine, 4-hydroxynonenal-lysine, and N(epsilon)-(carboxymethyl)lysine (CML), increased by approximately 3-fold, while levels of the protein oxidation marker, methionine sulfoxide (MetSO), were unchanged. Skin collagen was resistant to modification until the third week, when 2- to 3-fold increases in both CML and MetSO were observed. Our results document that iron overload increases lipid peroxidation, with concomitant increases in reactive aldehydes in plasma and chemical modification of tissue proteins. CML was a sensitive indicator of hepatocellular oxidative stress, compared to MetSO, while extensive modification of extracellular skin collagen was not observed until the late stages of iron overload and oxidative stress. These observations provide direct evidence for the contribution of reactive oxygen species, lipid peroxidation, and reactive carbonyl intermediates to the pathogenesis of iron-overload diseases.

Erythropoiesis: Comparison of Cytotoxic Aldehyde Generation in Beta-Thalassemia Patients Chelated with Deferoxamine or Deferiprone (L1) Versus NO Chelation.

The mechanism of iron-induced organ failure in iron overload disorders is not known, but it is conjectured that excess iron-catalyzed free radical generation contributes to organ damage. We hypothesized that free radical generation, quantified by the presence of 20 separate cytotoxic aldehydes in plasma, would be significantly increased in non-chelated beta-thalassemia major patients, in comparison to those chelated with either deferiprone (L1) or deferoxamine (desferal). We also report on red cell glutathione peroxidase activity in these patient groups, an enzyme involved in averting the damaging effects of free radicals. Ten patients were chelated with nightly subcutaneous infusions of desferal and 10 received the experimental oral chelator L1. Body iron burden was assessed by serum ferritin and hepatic iron concentrations. In comparison to non-chelated controls, significant decreases of 62% and 64% in total cytotoxic aldehyde concentrations were observed in patients chelated with desferal and L1, respectively (p < 0.001). Significantly lower red cell glutathione peroxidase activity was also observed in non-chelated controls, in comparison to those chelated with either desferal or L1 (p < 0.001). This is the first report on the concentrations of cytotoxic aldehydes in non-chelated beta-thalassemia major patients, and the first to report on the effects of L1 against cytotoxic aldehyde formation in plasma of patients with iron-overload.