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September 2009

Editor:
Michael Bissell, MD, PhD, MPH

Anti-Müllerian hormone as a serum marker
Genetic regulation of receptors for low-density lipoprotein cholesterol
Effect of free fatty acids and maternal triglycerides on placental lipoprotein lipase
D-lactate as an early marker for colon ischemia after open aortic reconstruction
Risk assessment of mercury exposure from gold mining

Anti-Müllerian hormone as a serum marker Anti-Müllerian hormone as a serum marker

The majority of treatment protocols for Hodgkin’s lymphoma consist of a combination of chemotherapy and radiotherapy. Using this strategy, childhood Hodgkin’s lymphoma (HL) has an event-free survival rate of approximately 90 to 95 percent and an overall survival rate of up to 96 percent. Because of these improved survival rates, long-term side effects are an important issue. Most studies report on late effects in adult HL patients, and little is known about the endocrine long-term effects in children treated for the disease. In adults, an important long-term effect of radiotherapy and chemotherapy is decreased gonadal function, especially after chemotherapy protocols containing high cumulative doses of potentially gonadotoxic agents, such as mechlorethamine and procarbazine. Gonadal function typically is measured in followup studies of long-term survivors of childhood cancer by analyzing luteinizing hormone (LH) and follicle-stimulating hormone (FSH). However, neither LH nor FSH reflects the ovarian reserve. In recent years, two new markers for ovarian function have become available—inhibin B and anti-Müllerian hormone (AMH). The authors conducted a study to evaluate the long-term effects of combination chemotherapy treatment for girls with HL on gonadal function using AMH and inhibin B as ovarian reserve parameters. LH, FSH, inhibin B, and AMH were measured in 32 women treated for pediatric HL with che-motherapy from 1974 to 1998, with the intention to avoid radiotherapy. All patients (median age, 25 years; range, 19.2–40.4 years) were in complete remission with a median followup time of 14 years (range, 5.7–24.5 years) after therapy. All patients were treated with combina-tion chemotherapy doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) or EBVD with or without mechlorethamine, vincristine, procarbazine, and prednisone (MOPP). Because of incomplete remission or relapse, involved field radiotherapy was needed for seven of 32 women. Results were compared with those for a healthy control group. The authors found that patients treated with six or more cycles of MOPP combination chemotherapy had significantly higher levels of FSH and lower serum levels of inhibin B and AMH compared with healthy women (FSH, 17.0 versus 6.0 U/L [P<.05]; inhibin B, 23.0 versus 112.5 ng/L [P<.01]; AMH, 0.39 versus 2.10 µg/L [P<.01]). AMH was also significantly lower in this group than in women whose treatment did not include MOPP (median, 0.39 versus 1.40 µg/L [P=.01]). The authors concluded that women treated during childhood for HL with MOPP seem to have a distinctly lower ovarian reserve, as measured by lower AMH values in early adulthood, than do healthy women. Moreover, AMH seems to be the only predictor that is sufficiently sensitive to detect this decrease in ovarian reserve.

Van Beek RD, van den Heuvel-Eibrink MM, Laven JSE, et al. Anti-Müllerian hormone is a sensitive serum marker for gonadal function in women treated for Hodgkin’s lymphoma during childhood. J Clin Endocrinol Metab. 2007;92:3869–3874.

Correspondence: Sabine M. P. F. de Muinck Keizer-Schrama at s.demuinckkeizer-schrama@erasmus.nl

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Genetic regulation of receptors for low-density lipoprotein cholesterol Genetic regulation of receptors for low-density lipoprotein cholesterol

The novel serum protease proprotein convertase subtilisin kexin type 9 (PCSK9) is gaining attention as a potential key regulator of serum low-density lipoprotein cholesterol. PCSK9 is a protease made by the liver and thought to degrade hepatic LDL receptors (LDLR). The mechanism by which PCSK9 degrades LDLR is not completely understood and is likely to be complex. When LDLR concentrations are decreased, the liver is less able to bind LDL from the circulation, and serum LDL cholesterol increases. As a result, gain-of-function and loss-of-function mutations in the PCSK9 gene have dramatic effects on serum LDLC concentrations in humans. Patients with activating mutations of PCSK9 have severe familial hypercholesterolemia and increased cardiovascular risk. These mutations in PCSK9 account for the approximately 10 to 25 percent of familial dominant hypercholesterolemia cases that cannot be explained by mutations affecting LDLR or apolipoprotein B. In contrast, heterozygous patients with loss-of-function mutations in PCSK9 have significantly decreased concentrations of LDL cholesterol in serum and dramatically decreased cardiovascular risk. Approximately two percent of African-Americans carry such mutations, with an accompanying 80 to 90 percent decreased risk of serious cardiovascular events. The first compound heterozygote for PCSK9 loss-of-function mutations recently was described. The patient, a healthy 32-year-old woman, had an extremely low serum LDL cholesterol of 140 mg/L (14 mg/dL) and no detectable PCSK9 protein as assessed by immunoprecipitation and Western blotting. These remarkable findings highlight the need for a robust and easy-to-perform immunoassay for measuring PCSK9 in human serum. Such an assay would allow comparison of LDLC and PCSK9 concentrations, shedding light on the importance of PCSK9 in regulating LDL cholesterol. Such an assay might also allow the medical field to predict which patients might best respond to a PCSK9 in-hibitor if one were to become available. The authors described, in detail, a dual monoclonal antibody sandwich ELISA to measure PCSK9 in human serum and correlate the measured PCSK9 concentrations with LDL and total cholesterol concentrations. They used recombinant human PCSK9 pro-tein and two different anti-PCSK9 monoclonal antibodies to build the sandwich ELISA. The authors also measured PCSK9 and lipids in 55 human serum samples and correlated the results. They used the anti-PCSK9 antibodies to assay lipoprotein particle fractions separated by sequential flotation ultracentrifugation. The authors found that serum concentrations of PCSK9 ranged from 11 to 115 µg/L and were directly correlated with serum concentrations of LDL cholesterol (r=0.45; P=.001) and total cholesterol (r=0.50; P=.0003) but not with triglycerides (r=0.15; P=.28) or high-density lipoprotein cholesterol concentrations (r=0.13; P=.36). PCSK9 was not detectable in any lipoprotein particle fraction, including LDL. The au-thors concluded that PCSK9 is present in human serum but likely is not associated with specific lipoprotein particles. The circulating concentrations of human PCSK9 are directly correlated with LDL and total cholesterol concentrations.

Alborn W, Cao G, Careskey HE, et al. Serum proprotein convertase subtilisin kexin type 9 is correlated directly with serum LDL cholesterol. Clin Chem. 2007;53:1814–1819.

Correspondence: Robert J. Konrad at konrad_robert@lilly.com

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Effect of free fatty acids and maternal triglycerides on placental lipoprotein lipase Effect of free fatty acids and maternal triglycerides on placental lipoprotein lipase

Fetal demand for fatty acids is high during the last trimester of pregnancy as fatty acids are critical for normal neural and vascular development. The fetus is able to synthesize saturated and monounsaturated fatty acids from glucose and ketone bodies but depends entirely on placental transport for its supply of the essential fatty acids linoleic and α-linolenic acid. The essential fatty acids and their derivatives, the long-chain polyunsaturated free fatty acids (LC-PUFAs) arachidonic (ARA) and docosahexaoneic (DHA), are necessary constituents of membranes, act as precursors of cellular signaling molecules, and are particularly important for the developing brain and retina. Triglycerides are transported in the blood-stream as lipoproteins and must be hydrolyzed into free fatty acids before they can be transferred across the syncytiotrophoblast of the human placenta. Maternal hypertriglyceridemia is a normal condition in late gestation and ensures adequate nutrient supply to the fetus. Placental lipoprotein lipase (LPL) is involved in the initial step in transplacental fatty acid transport as it hydrolyzes maternal triglycerides to release free fatty acids. The authors investigated LPL activity and protein (Western blot) and mRNA expression (real-time RT-PCR) in the placenta of an LPL-deficient mother with marked hypertriglyceridemia. They found that the LPL activity was fourfold lower, LPL protein expression 50 percent lower, and mRNA expression threefold higher than that of normal, healthy placentas at term (n=4–7). To further investigate the role of maternal lipids in placental LPL regulation, the authors isolated placental cytotrophoblasts from term placentas and studied LPL activity and protein and mRNA expression after incubation in Intralipid (as a source of triglycerides) and oleic acid, linoleic acid, and a combination of oleic, linoleic, and arachidonic acids, as well as insulin. Intralipid (40 and 400 mg/dL) decreased LPL activity by approximately 30 percent (n=10–14; P<.05), and 400 µM linoleic and linoleicoleicarachidonic acid (n=10) decreased LPL activity by 37 and 34 percent, respectively. No major changes were observed in LPL protein or mRNA expression. The authors found that insulin had no effect on LPL activity or protein expression in the cultured trophoblasts. They concluded that the activity of placental LPL is reduced by high levels of maternal triglycerides or free fatty acids, or both. This regulatory mechanism may serve to counteract an excessive delivery of free fatty acids to the fetus in conditions where maternal triglyceride levels are markedly increased.

Magnusson-Olsson AL, Lager S, Jacobsson B, et al. Effect of maternal triglycerides and free fatty acids on placental LPL in cultured primary trophoblast cells and in a case of maternal LPL deficiency. Am J Physiol Endocrinol Metab. 2007;293:E24–E30.

Correspondence: A. L. Magnusson-Olsson at anneliese.olsson@fysiologi.gu.se

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D-lactate as an early marker for colon ischemia after open aortic reconstruction D-lactate as an early marker for colon ischemia after open aortic reconstruction

The reported incidence of colon ischemia after open aortic surgery for infrarenal aortic aneurysm varies between three percent for elective surgery and 36 percent for emergency surgery, with mortality rates of up to 90 percent. Many factors have been associated with asymptomatic and symptomatic colon ischemia, which predominantly affects the sigmoid colon. Colon ischemia can be caused by hypoperfusion, embolization, and reperfusion injuries. Early detection of this condition is essential to ensure rapid and correct treatment. Yet laboratory parameters with acceptable sensitivity and specificity, or other tools for early detection, have not proven helpful in diagnosing intestinal ischemia in routine clinical practice. Based on the concept that breakdown of mucosal barrier function due to intestinal hypoperfusion is the earliest dysfunction of ischemic colitis, detection of translocation parameters might allow prompt diagnosis of early tissue necrosis. Two promising analytes are D-lactate and endotoxins (lipopolysaccharides, or LPS), which are metabolic products or components of the normal anaerobic bacteria of the gastrointestinal tract. Plasma D-lactate levels have been reported to be increased early after the damage to the intestinal mucosal barrier in animal models. Plasma D-lactate was also elevated in patients with colon ischemia in a retrospective study. LPS, major constituents of the outer membrane of Gramnegative bacteria, are released from bacteria when replicating, dying, or lysing. Increased LPS levels have been related to impaired mucosal barrier. However, contradictory evidence concerning the association between circulating endotoxin levels and ischemia-induced intestinal injury has been reported. The authors conducted a prospective pilot cohort study to assess the clinical significance of D-lactate and LPS as early markers for ischemic colitis proven by histology. For the study, 12 consecutive patients underwent surgery between February and April 2003. Six patients underwent emergency surgery and six patients elective aortic surgery. D-lactate and endotoxin levels were measured in blood samples collected according to a standardized protocol. For histological examination, biopsies were obtained by sigmoidoscopy on days four through six after surgery or earlier if indicated clinically. The authors found that as early as two hours postoperatively, elevated plasma levels of D-lactate were measured in patients with histologi-cally proven ischemic colitis. The peak of D-lactate elevation was on postoperative days one and two. Concentration of plasma endotoxin was not significantly different in patients with or without ischemic colitis. The authors’ data suggest that plasma D-lactate levels are a useful marker for early detection of ischemic colitis secondary to aortic surgery.

Assadian A, Assadian Q, Senekowitsch C, et al. Plasma D-lactate as a potential early marker for colon ischemia after open aortic reconstruction. Eur J Vasc Endovasc Surg. 2006;31:470–474.

Correspondence: Afshin Assadian, MD, at afshin_assadian@yahoo.de

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Risk assessment of mercury exposure from gold mining Risk assessment of mercury exposure from gold mining

Mercury is a well-known pollutant of terrestrial and aquatic ecosystems that is damaging human health and the environment. Mercury continues to be used in gold mining in Suriname to separate small amounts of gold flakes from large volumes of soil and sediment. Each year, gold mining leads to 10 to 60 tons of mercury (Hg) being released in Suriname’s interior rainforest. All of the mercury used in gold mining is released into the environment, affecting more than 50 Suriname villages housing approximately 50,000 indigenous and tribal residents, which is 10 percent of the country’s population. Once released, elemental and inorganic mercury are converted to the highly toxic methyl-mercury form and bioaccumulate in the aquatic food chain. The authors conducted a project to support the indigenous people in Kwakoegron, Suriname, in self-diagnosing public and environmental health problems. The specific objectives, defined by the people of Kwakoegron, were to determine for themselves if they are at risk of exposure to mercury contamination, measure the extent of the contamination problem, and initiate an intervention plan. Field work was conducted from June 2005 to April 2006. Community members were trained to collect hair samples for analysis using methods designed to maximize sample quality and consistency and minimize cross-contamination. Each hair sample, of approximately 20 mg, was weighed, added to the sample boat, and analyzed immediately without preservation or storage. Technicians educated in analytical chemistry and trained in the operation of the portable Lumex Zeeman Hg analyzer measured the total mercury (THg) for each hair sample. The technicians found that hair samples from 16 of the 22 participants had mercury levels of 2.2 to 20.2 µg/g THg, exceeding normal (2 µg/g) THg levels for hair. During confidential, individual meetings and a followup community meeting, information was shared regarding the mercury levels found, what the numbers meant scientifically, the potential health effects, and how exposure levels might be reduced. At the conclusion of the followup meeting, the Kwakoegron community proposed an intervention plan that had as its principle parts routine analysis of mercury exposure to monitor trends and track the effects of expo-sure-reduction efforts; routine health assessments to determine the effects of mercury exposure, particularly in children younger than five years of age; and fish advisories based on fish biology and trophic level or on the specific measurement of mercury levels in various fish species from as-sorted locations and at established times during the year. The authors concluded that this project showed that a democratic approach to science does not automatically compromise the orderly search for answers. Their experience in Kwakoegron suggests that the collaborative relationship that emerges by empowering an indigenous community to initiate its own research projects and address the needs it identifies can contribute to the risk-assessment process. The project also showed that when Kwakoegron was acknowledged as an equal partner, the risk-assessment process led to an open exchange of information and an intervention plan that was pragmatic and acceptable in the context of the community’s unique social and cultural needs.

Peplow D, Augustine S. Community-directed risk assessment of mercury exposure from gold mining in Suriname. Pan Am J Public Health. 2007;22:202–210.

Correspondence: Daniel Peplow at dpeplow@u.washington.edu

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Dr. Bissell is professor, Department of Pathology, Ohio State University, Columbus.