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June 2010

Editor:
Michael Bissell, MD, PhD, MPH

Iodine and magnesium levels in pre-eclampsia Iodine and magnesium levels in pre-eclampsia

Pre-eclampsia, which remains a leading cause of maternal and neonatal mortality and morbidity, is usually diagnosed in late pregnancy in women with increased blood pressure and proteinuria. The symptoms disappear shortly after a woman delivers the placenta after giving birth. Although the causes of the symptoms are unknown, studies have shown that pre-eclampsia is a multisystemic disease affecting the placental endothelium, with an unidentified circulating factor, or factors, altering vascular function. It has been reported that pre-eclamptic women have pathologically low levels of circulating magnesium compared with healthy pregnant women. Moreover, magnesium assimilation is defective when iodine is missing. Iodine is an essential component of thyroid hormones. The thyroid gland shares its capacity to actively accumulate io-dine with several other tissues, including stomach, salivary glands, and lactating mammary glands. The amount of iodine in the human body ranges from about 30 to 50 mg, and less than 30 percent is present in the thyroid gland. About 60 to 80 percent of total iodine is nonhormonal and concentrated in tissues other than the thyroid gland, but the biologic role of nonhormonal iodine is unknown. Geographic differences in the rates of breast, endometrial, and ovarian cancer appear to be inversely correlated with dietary iodine intake. Endocrinologic considerations sug-gest that a low dietary iodine intake may increase effective gonadotropin stimulation, which in turn may produce a hyperestrogenic state char-acterized by a relatively high production of estrone and estradiol and a relatively low estriol to estrone-plus-estradiol ratio. This altered endo-crine state may increase the risk of breast, endometrial, and ovarian cancers. Increasing dietary iodine intake may reduce the risk of these can-cers. Turkey has been included in the countries with endemic goiter. Studies support the assertion that iodine deficiency is endemic in the northeastern Anatolia region of Turkey, including the city of Erzurum. The authors conducted a study to investigate the differences in blood magnesium concentration and placental tissue iodine concentration in women with severe pre-eclampsia and healthy pregnant women in Erzu-rum. They obtained placental tissue and blood specimens from 20 severely pre-eclamptic and 15 healthy pregnant women. Iodine levels in pla-cental tissue were determined by the Foss method based on the Sandell-Kolt-hoff reaction. The authors found that placental tissue iodine levels were lower in women with severe pre-eclampsia than in healthy pregnant women (4.30±1.36 ng of iodine/mg protein versus 7.71±2.84 ng of iodine/mg tissue protein; P<001), as were blood magnesium levels (1.63±0.05 mg/dL versus 1.87±0.05 mg/dL; P<.001). A positive correlation was noted between placental tissue iodine levels and blood magnesium levels in women with severe pre-eclampsia (r=0.55; P<.05), but no such correlation was observed in healthy pregnant women (r=0.23; P=.41). The authors concluded that magnesium assimilation is known to be defec-tive when iodine levels are insufficient. In northeast Anatolia, where iodine deficiency is common, clinical trials of iodine supplementation should be considered for pre-eclamptic therapy.

Gulaboglu M, Borekci B, Halici Z. Placental tissue iodine level and blood magnesium concentration in pre-eclamptic and normal pregnancy. Int J Gyn Obstet. 2007;98:100–104.

Correspondence: M. Gulaboglu at gulaboglumine@yahoo.com

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Age-related mitochondrial DNA point mutations in people with mitochondrial myopathy Age-related mitochondrial DNA point mutations in people with mitochondrial myopathy

Since discovering the complete sequence of human mitochondrial DNA, numerous mutations have been described, including large-scale re-arrangements and point mutations. Mitochondrial DNA (mt-DNA) mutations typically are associated with diseases affecting the oxidative phosphorylation system, called mitochondrial diseases. They are usually found in a heteroplasmic state, coexisting with mutant and wild type mtDNA, with high proportions of mutant mtDNA. The same mutations associated with mitochondrial diseases have also been found in normal aged tissues. However, unlike with mitochondrial diseases, age-related mtDNA deletions are found in very low proportions—usually less than one percent of total mtDNA—and can only be detected by sensitive methods, such as polymerase chain reaction. As with mt-DNA deletions, small duplications of the mtDNA control region were found in aged tissues in very low levels. The free radical theory is a popular theory pro-posed to explain the aging process because free radical reactions may be associated with genetic and environmental factors and neurodegenera-tive diseases. The mitochondrial respiratory chain is considered the main source of oxygen radicals in the cell, and due to the proximity of mtDNA, located in the inner mitochondrial membrane, age-related mtDNA mutations are thought to be caused by oxidative damage. A vicious cycle might contribute to the aging process: Electron transport chain normally would generate free radicals that would induce oxidative damage to proteins, lipids, and mtDNA in mitochondria. Consequently, a respiratory chain defect would develop, leading to an increase in free radical production by the electron transport chain. The vicious cycle would cause an exponential increase in mtDNA mutations with time, leading to aging and age-related neurodegenerative diseases. Although very popular, there is no strong evidence confirming the existence of this vicious cycle, and the significance of these mutations to the aging process is unclear. Several point mutations in the control region (D-loop) of mt-DNA were found to accumulate in aged tissues and could indicate that specific mutations in this important region of mtDNA would play a more im-portant role in the aging process. Two mutations—A189G and T408A—were found to be specific to skeletal muscle and were present in higher proportions (up to 60 percent) than the other age-related mutations described previously. The authors conducted a study to verify whether pa-tients with a mitochondrial disease are more predisposed to accumulate the A189G and T408A mutations in the D-loop and to confirm their age-associated nature. The authors evaluated the presence and levels of heteroplasmy of these two mutations in the muscle DNA of 52 indi-viduals of different ages (21 age-matched controls and 31 patients with single or multiple mtDNA deletions). The frequency of both mutations was significantly increased with age, but no differences were observed between the group of patients and their age-matched controls. The au-thors did not observe a correlation between levels of heteroplasmy and age. The results confirm the age-related nature of the A189G and T408A mutations in the D-loop in controls and patients with mitochondrial disease but do not suggest that patients are more predisposed to develop-ing age-related point mutations.

Da Costa CK, Kiyomoto BH, Schmidt B, et al. Age-related mitochondrial DNA point mutations in patients with mitochondrial myopathy. J Neurol Sci. 2007;263:139–144.

Correspondence: R. Pedro de Toledo at chtengan@neuro.epm.br

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Heritability of testosterone levels in adult twins Heritability of testosterone levels in adult twins

The testis is the target organ of the hypothalamic-pituitary-gonadal axis. Gonadotropin-releasing hormone secreted by the hypothalamic pulse generator is released in a pulsatile fashion into the portal vascular system, through which the hypothalamus communicates with the pitui-tary gland. This pulsatile secretion is how the anterior pituitary releases follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The main functions of the human testis are to produce hormones and regulate spermatogenesis. FSH released through the pituitary gland stimulates testicular Sertoli cells to produce inhibin B, which has been shown to be a serum marker of Sertoli cell function—that is, spermatogenesis—in adult males. FSH release and synthesis consist of a GnRH dependent and an autonomous component that is inhibited by inhibin B. Reproduc-tive hormones have been compared in monozygotic and dizygotic male twins, and inhibin B levels have been reported to be elevated in dizy-gotic twins. No such difference was found for FSH. This led to the suggestion that the FSH-inhibin B endocrine feedback axis functions differ-ently in male dizygotic twins. However, because non-twin siblings were not included in these analyses, it is unclear whether dizygotic twins have significantly higher inhibin B levels than usual and whether monozygotic twins have significantly lower inhibin B levels. Testosterone level has been regarded as highly heritable, especially in young adult males, with an estimated 66 percent of the variance due to genetic compo-nents. Serum hormone concentrations are subject to regulation at the genetic level and have environmental influences. The authors conducted a study to evaluate the genetic contribution of the various endocrine components of the hypothalamic-pituitary-testicular axis. They assayed a sample of adult male twins and their siblings for plasma hormones, including inhibin B, LH, FSH, sex hormone-binding globulin (SHBG), and testosterone. They conducted assays on 128 adult males—20 monozygotic twin pairs, seven single monozygotic twins, 10 dizygotic twin pairs, 27 single dizygotic twins, and 34 siblings of twins, constituting 10 sibling pairs—aged 15.6 to 68.7 years. The authors chose males as study sub-jects because they provide a relatively stable endocrine environment compared to females due to the absence of a monthly menstrual cycle. Fur-thermore, the authors aimed to verify the earlier reported difference between monozygotic and dizygotic twins with regard to inhibin B secre-tion. The authors included the siblings of the monozygotic and dizygotic twins in their analyses to test for the possibility that twins differ from singleton offspring. The study compared hormone levels across zygosity groups and obtained heritability estimates using maximum likelihood variance component analysis. Heritability estimates ranged from 56 percent (testosterone) to 81 percent (inhibin B and SHBG). For LH and FSH, the heritability was estimated at 68 percent and 80 percent, respectively. No mean differences in hormone levels were observed across groups. The authors concluded that all measured hormones are highly heritable. They could not confirm a difference in the FSH-inhibin B feedback sys-tem between dizygotic twin males and monozygotic twin males.

Kuijper EAM, Lambalk CB, Boomsma DI, et al. Heritability of reproductive hormones in adult male twins. Hum Reprod. 2007;22(8): 2153–2159.

Correspondence: E. A. M. Kuijper at e.kuijper@vumc.nl

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Drug susceptibility in hepatitis B virus populations Drug susceptibility in hepatitis B virus populations

The availability of oral nucleoside/nucleotide analog anti-hepatitis B virus reverse-transcriptase inhibitors has greatly improved the man-agement of patients with chronic hepatitis B. Four nucleoside/nucleotide analog hepatitis B virus (HBV) reverse-transcriptase (RT) inhibitors—lamivudine, adefovir dipivoxil, entecavir, and telbivudine—are approved in the United States for treating chronic hepatitis B. Due to the persis-tent nature of chronic HBV infection, largely attributable to the stability of HBV covalently closed circular DNA, these therapies rarely produce HBsAg seroconversion and therefore require prolonged administration to control disease in most patients. However, long-term therapy can be associated with the emergence of resistant HBV strains, leading to loss of therapeutic benefit and resumption of liver disease progression. Resis-tance to lamivudine results from the selection of HBV RT YMDD mutations—rtM204V and rtM2041—and occurs in about 20 percent of patients per year of treatment. Lamivudine-resistance mutations confer cross-resistance to other L-nucleoside analogs, such as telbivudine, clevudine, and emtricitabine, and contribute to resistance to entecavir. In contrast, adefovir dipivoxil maintains clinical efficacy against lamivudine-resistance mutations, but its long-term administration selects for the resistance mutation rtN236T or rtA181V, or both, although at much lower incidence than that in lamivudine therapy. The expanding use and prolonged administration of approved HBV RT inhibitors, as well as the de-velopment of new agents, place an increasing emphasis on monitoring and identifying new drug-resistance mutations in antiviral therapy. Evaluation of the in vitro drug susceptibilities of resistance-associated mutations is a crucial component of any resistance surveillance program. A novel plasmid expression vector for cloning the entire HBV genome has been created to facilitate the expression of full-length HBV clinical isolates that were amplified with a pair of primers encompassing a highly conserved region in the HBV genome. The cloned clinical isolates could then be transfected into hepatoma cell lines, and in vitro drug susceptibilities could be tested. Because of the quasispecies nature of HBV, and because the assay was based on testing individual clones of clinical isolates, different isolates showed large variations in replication capaci-ties, even among those from the same serum collections. The authors developed a procedure for cloning serum HBV quasispecies populations and for phenotypic analysis of the cloned populations for in vitro drug susceptibility. They compared equivalent sequences to the respective serum HBV DNAs of the cloned quasispecies by population sequencing. Analysis of individual clones revealed that each population contained a diversity of HBV quasispecies. Furthermore, secreted HBV in the supernatant following transfection of the quasispecies populations remained mostly unchanged from the respective input populations. The authors also tested HBV obtained from patients who had developed resistance to adefovir or lamivudine, as demonstrated by development of the rtA181V or rtL180M/M204V mutations in HBV polymerase, respectively. Phe-notypic analysis demonstrated that a population containing the HBV rtA181V mutation showed a 2.9-fold increase in the 50 percent effective concentration (EC50) for adefovir compared to the wild-type baseline isolate, while the lamivudine-resistant HBV quasispecies population showed a greater than 1,000-fold increase in the lamivudine EC50. In summary, the authors developed a strategy for cloning full genome HBV quasispecies populations from patient sera, which could provide a useful tool in clinical HBV drug-resistance phenotyping and studies of the evolution of clinical viral species.

Zhu Y, Curtis M, Snow-Lampart A, et al. In vitro drug susceptibility analysis of hepatitis B virus clinical quasispecies populations. J Clin Microbiol. 2007;45:3335–3341.

Correspondence: Yuao Zhu at yuao.zhu@gilead.com

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Metabolic syndrome in endocrinological context Metabolic syndrome in endocrinological context

Adult Treatment Panel III guidelines define metabolic syndrome as the presence of three of five determinants—abdominal adiposity, hyper-tension, low high-density lipoprotein cholesterol, elevated triglyceride levels, and abnormal fasting plasma glucose. The authors previously re-ported that adding elevated two-hour plasma postglucose challenge levels (2hPG) as a criterion significantly increased the prevalence of meta-bolic syndrome in a cohort of community-dwelling Caucasian adult participants in the Baltimore Longitudinal Study of Aging (BLSA). Their results showed that the prevalence of the syndrome was higher in men compared with women, which may be related to differences in andro-genic effects. It has been reported that lower total testosterone and sex hormone-binding globulin (SHBG) levels predicted subsequent devel-opment of type 2 diabetes mellitus. Baseline total testosterone and SHBG were significantly lower in men who subsequently developed type 2 diabetes. Another study reported that total testosterone and SHBG predict the development of metabolic syndrome and type 2 diabetes in mid-dle-aged men. It was also reported that baseline androgen levels were inversely correlated with developing the syndrome. The authors con-ducted a study to determine the relationship between metabolic syndrome and circulating androgen levels in a cohort of men in the BLSA. Study participants were Caucasian community-dwelling men in the BLSA who underwent a fasting two-hour oral glucose tolerance test and had serum concentrations of total testosterone, dehydroepiandrosterone sulfate, and SHBG levels measured. The prevalence of metabolic syn-drome was 4 percent, 21 percent, 21 percent, and 18 percent for men between the ages of 20 and 39, 40 and 59, 60 and 79, and 80 and 94 years, respectively. Total testosterone and SHBG were inversely related to developing the syndrome during a mean followup period of 5.8 years (range, 1.5–14 years), whereas the free T index and body mass index were positively related to incidence of the syndrome. Age alone did not predict the development of metabolic syndrome, nor did including abnormal two-hour plasma postglucose challenge levels in the classification of metabolic syndrome. Stepwise proportional hazards regression analyses showed that among the various measurements, SHBG levels exerted the greatest influence on developing the syndrome. The authors concluded that the prevalence of metabolic syndrome increased with age, and this was associated with lower androgen levels. Lower total testosterone and SHBG predicted a higher incidence of metabolic syndrome.

Rodriguez A, Muller DC, Metter EJ, et al. Aging, androgens, and the metabolic syndrome in a longitudinal study of aging. J Clin Endocrinol Metab. 2007;92(9):3568–3572.

Correspondence: Dr. Annabelle Rodriguez at adrodrig5@jhmi.edu

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Detecting hepatitis B virus and anti-HBV in blood donors Detecting hepatitis B virus and anti-HBV in blood donors

The World Health Organization estimates that 2 billion people have been infected with hepatitis B virus, of whom 350 million are chronically infected. Approximately 1 million people die each year from this infection, and 4 million new infections occur annually. Fourteen to 40 percent of chronic carriers develop liver cirrhosis, and more than 500,000 new hepatocellular carcinoma cases are diagnosed each year. The introduction of hepatitis B surface antigen (HBsAg) screening reduced, but did not eliminate, the risk of hepatitis transfusion-transmitted infections. It has been demonstrated that the frequency and detection of hepatitis B virus (HBV) DNA at a low viremia does not correlate with serologic markers in chronic HBV carriers. One of the disadvantages of anti-HBc screening is the permanent deferral of donors with false-positive anti-HBc results due to the lack of a confirmatory test. Since the introduction of commercially available routine anti-HBc testing at Canada’s Hema-Quebec blood center in April 2003, approximately 0.5 percent of donors have been deferred on the basis of reactive anti-HBc results. To evaluate the number of infectious donors intercepted by the assay and the difference between antibody to hepatitis B surface antigen (HBs)-positive and -negative do-nors in terms of viremia, the authors gathered HBV DNA in anti-HBc reactive samples. They tested 1,169 reactive donations collected between October 2004 and November 2005 using their in-house HBV nucleic acid testing (NAT) assay, which is a conventional polymerase chain-reaction test for amplifying part of the viral S gene. HBV DNA-positive samples were further investigated by sequencing to evaluate the genetic diversity of the virus in Quebec’s blood donor population. The authors found that all HBsAg-positive samples were detected by the NAT assay. Overall, 38 (3.25 percent) anti-HBc-positive samples were found to be positive for the presence of HBV DNA. Of these 38, 12 donations with a low level of HBV DNA were HBsAg negative. The sequencing results showed various genotypes and subtypes within the same genotype. The authors concluded that the 3.25 percent HBV DNA-positivity rate among the anti-HBc-reactive donations, and more particularly the low level of HBV DNA observed in occult donations, emphasizes the importance of using a sensitive assay to detect HBV DNA in conjunction with other markers. The HBV genetic diversity found in this donor population reflects the province’s demographics, particularly in the Montreal area, where most of the positive donors were from.

Chevrier MC, St-Louis M, Perreault J, et al. Detection and characterization of hepatitis B virus of anti-hepatitis B core antigen-reactive blood donors in Quebec with an in-house nucleic acid testing assay. Transfusion. 2007;47:1794–1802.

Correspondence: Marie-Claire Chevrier at marieclaire.chevrier@hema-quebec.qc.ca

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Hereditary hemochromatosis in a Turkish population Hereditary hemochromatosis in a Turkish population

Hereditary hemochromatosis is an autosomal recessive disorder characterized by iron overload. It is associated with mutations of the HFE gene, which is located on the short arm of chromosome 6. Hereditary hemochromatosis (HH) is the most common genetic disorder seen in Cau-casians in the United States, Europe, and Australia, with a rate of two to five per 1,000 people. Increased transferrin saturation was used for screening before HFE gene mutation was established. Iron accumulates in the liver, pancreas, joints, heart, hypophysis, and skin due to the in-crease in intestinal absorption. Iron overload causes hepatocellular damage and activates hepatic stellate cells, after which fibrosis develops. The gene responsible for HH was defined in 1996 and named HFE. C282Y and H63D mutations were described in the HFE gene. The mutations were detected in 65 to 100 percent of cases that had clinical features of HH. Determination of HFE gene mutation confirms the diagnosis and also enables diagnosis of the disease in the asymptomatic period. Therefore, HFE gene mutation screening and transferrin saturation are impor-tant for early diagnosis in the relatives of people diagnosed with hemochromatosis. Iron overload is seen in some chronic liver disease patients, related to various etiologies, but it is not observed in others. HFE gene mutations may be responsible for this discrepancy. In Turkey, there have been limited studies of HFE gene mutations. In these studies, C282Y mutation was not found in healthy donors or HH patients. On the other hand, the impact of the H63D mutation, which was found in Turkey, is not known. The authors conducted a study in which they analyzed the C282Y and H63D gene mutations in healthy control subjects and chronic liver disease patients with and without iron overload to investigate the relationship between iron overload and HFE gene mutation in chronic liver disease patients. They evaluated 113 chronic liver disease patients and 138 healthy controls regarding their clinical, biochemical, and genetic parameters. Each group was divided into two subgroups—those with 45 percent transferrin saturation and those with transferrin saturation greater than 45 percent. HFE gene mutation was analyzed by the poly-merase chain reaction–restriction fragment length polymorphism method. C282Y homozygote, heterozygote, and wild-type mutation rates were 1.7, 0, and 98.3 percent in patients, and 0, 1.4, and 98.6 percent in controls, respectively. H63D homozygote, heterozygote, and wild-type muta-tion rates were 1.8, 24.7, and 73.5 percent in patients, and 1.4 , 24, and 74.6 percent in controls, respectively. Mutation rates were not statistically different in patients with high and normal transferrin saturation. Iron overload was positively correlated with biochemical activity and Child-Pugh score (P<.05). In multivariate analysis, H63D homozygotic mutation was an independent factor for developing hepatocellular carcinoma (P=.004). The authors concluded that C282Y mutation is very rare in Turkey. Iron overload is not related to H63D mutation but is positively cor-related with biochemical activity and Child-Pugh score in chronic diseases.

Yonal O, Hatirnaz O, Akyuz F, et al. HFE gene mutation, chronic liver disease, and iron overload in Turkey. Dig Dis Sci. 2007; 52: 3298– 3302.

Correspondence: Z. Mungan at munganz@istanbul.edu.tr

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Relationship between C-reactive protein and atherosclerosis in young people Relationship between C-reactive protein and atherosclerosis in young people

The relationship between inflammatory factors and coronary heart disease suggests that subclinical chronic inflammation may play a major role in the development of atherosclerosis. Although the relationship between C-reactive protein (CRP) and the risk of coronary heart disease (CHD) has been shown in adults, limited studies have been conducted among young people. Some studies documented a correlation between serum CRP and white blood cell count and some CHD risk factors among youths. The serum concentration of CRP is known to be an independ-ent risk factor and predictor for CHD. Oxidative stress may have proinflammatory effects, but data about its relationship with CRP in healthy individuals are limited. In a previous study, the authors found a higher level of oxidative stress in the offspring of parents with premature myo-cardial infarction than in control subjects. Oxidative stress may be a determinant of CRP concentrations and promotes the proatherosclerotic in-flammatory process. The authors conducted the study reported herein to investigate the association of serum CRP with generalized and ab-dominal obesity, as well as body fat composition, blood pressure, lipid profile, and the oxidative stress markers malondialdehyde (MDA) and conjugated diene (CDE) among children and adolescents. For their population-based study of 512 young people, aged 10 to 18 years, they ob-tained anthropometric and blood pressure measurements and quantified fasting blood sugar, total cholesterol, high-density lipoprotein choles-terol, triglycerides, CRP, MDA, and CDE. They also calculated low-density lipoprotein cholesterol for samples with triglycerides of not more than 4.52 mmol/L. The authors found that mean trigly-cerides, waist and hip circumferences, percentage body fat, subcutaneous fat, and sys-tolic blood pressure increased significantly with increasing body mass index. In con-trast, the mean LDL and total cholesterol were higher in underweight than normal weight individuals and then increased significantly from normal to higher body mass index categories. Mean HDL cholesterol significantly decreased with increasing body mass index. Overall, CRP, MDA, and CDE were significantly correlated with measures of abdominal obesity. Serum CRP, MDA, and CDE significantly increased in the upper quartiles of waist circumference. Study participants with higher CRP concentrations were more likely to have metabolic syndrome and high oxidative stress markers. The authors concluded that their findings indicate a significant positive association between CRP and oxidative stress markers in healthy young people, as well as an increase in these markers in the upper quartiles of waist circumference, but not body mass index. Oxidative stress and CRP may interact in the early in-flammatory processes of atherosclerosis.

Kelishadi R, Sharifi M, Khosravi A, et al. Relationship between C-reactive protein and atherosclerotic risk factors and oxidative stress markers among young persons 10-18 years old. Clin Chem. 2007;53:456–464.

Correspondence: Dr. Roya Kelishadi at kelishadi@med.mui.ac.ir

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.