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December 2006

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Michael Bissell, MD, PhD, MPH

Pharmacogenetics of amitriptyline therapy
Coagulation markers in cancer patients
Performance characteristics of automated natriuretic peptide assays
Factors that influence month-of-birth pattern in narcolepsy
Urinary excretion of homocysteine-thiolactone
Correlation between thromboxane A2 and in vitro aggregation in platelets

bullet Pharmacogenetics of amitriptyline therapy

Tricyclic antidepressants, in particular amitriptyline, have been the cornerstones of antidepressive therapy for more than three decades. Treatment guidelines recommend using tricyclic antidepressants (TCAs) only in patients with psychotic features and treatment resistance. The major pathway of amitriptyline (AT) metabolism is demethylation to nortriptyline (NT), mainly by cytochrome P450 2C19 (CYP2C19). NT is an active compound, which is why the sum of both concentrations is used to guide therapy in therapeutic drug monitoring. NT is hydroxylated by cytochrome P450 2D6 (CYP2D6), forming the inactive metabolite 10-0H-NT. The authors conducted a prospective blinded study of depressive Caucasian hospital inpatients treated with AT. They determined the CYP2C19 and CYP2D6 genotypes, measured serum concentrations of AT and NT, and sought correlations with adverse events and therapy response. Fifty Caucasian inpatients with at least medium-grade depressive disorder received amitriptyline at a fixed dose of 75 mg twice a day. Blood samples for concentration monitoring of amitriptyline and nortriptyline were taken weekly until discharge, along with evaluations of depression (Hamilton Depression Scale and Clinical Global Impression Scale) and side effects (Dosage Record and Treatment Emergent Symptoms Scale; DOTES) scores. In a receiver operating characteristic analysis, nortriptyline, but not amitriptyline, concentrations correlated with side effects (DOTES sum score, =5; area under the curve, 0.733; P=0.008). Carriers of two functional CYP2D6 alleles had a significantly lower risk of side effects than carriers of only one functional allele (12.1 versus 76.5 percent; P=0.00001). The lowest risk was observed for carriers of two functional CYP2D6 alleles combined with only one functional CYP2C19 allele (0 of 13 versus 9 of 11 [81.8 percent] for the high-risk group; P=0.00004). The authors found no correlations between drug concentrations or genotypes and therapeutic response. They concluded that combining pharmacogenetic testing for CYP2D6 and CYP2C19 identifies patients with low risk for side effects in amitriptyline therapy and could possibly be used to individualize antidepressive regimens and reduce treatment costs. Identifying genotypes associated with slightly reduced intermediate metabolism may be more important than anticipated. It could also be the key to demonstrating cost-effectiveness for CYP2D6 genotyping in critical dose drugs.

Steimer W, Zopf K, Von Amelunxen S, et al. Amitriptyline or not, that is the question: Pharmacogenetic testing of CYP2D6 and CYP2C19 identifies patients with low or high risk for side effects in amitriptyline therapy. Clin Chem. 2005;51:376385.

Werner Steimer, Institut fur Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universitat Munchen, Ismaningerstrasse 22, D-81675 Munich, Germany;

bullet Coagulation markers in cancer patients

It is estimated that venous thromboembolic disease occurs in approximately 6.8 percent of patients with an underlying malignancy. A constellation of factors contributes to the hypercoagulable state of cancer. Chemotherapy, surgery, immobilization, and comorbid conditions are associated with increased risk of clotting in these patients. The authors prospectively assessed the use of oral anticoagulation in patients with malignancy to evaluate the utility of certain hemostatic markers in patients undergoing oral anticoagulation. Patients (n=223) with a first episode of venous thromboembolic disease received oral anticoagulation with warfarin for a target international normalized ratio of 2 to 3. Plasma coagulation markers were measured before instituting warfarin and at three monthly intervals thereafter. The median duration of oral anticoagulation was 6.7 months (range, two weeks to 11 months). Major bleeding episodes occurred in 18 patients (eight percent), and minor hemorrhagic events occurred in 15 (6.7 percent) patients. Patients with advanced malignancy (P=0.032), history of surgery (P=0.057), and poor performance status (P=0.001) were more likely to experience major bleeding episodes. Recurrence of venous thromboembolic disease was diagnosed in 31 patients (14 percent). At univariate analysis, advanced stage of cancer (P=0.03), performance status greater than one (P=0.001), treatment with chemotherapy (P=0.01), metastatic liver disease (P=0.03), higher D-dimer (P=0.001), and thrombin antithrombin complex levels (P=0.01) were predictive of recurrent venous thromboembolic disease. At multivariate analysis, poor performance status (P=0.01) and D-dimer levels (P=0.001) predicted recurrent venous thromboembolic disease. Persistent activation of coagulation, as indicated by an upward trend in D-dimer (P=0.001) and antithrombin (P=0.001), was observed in patients who developed recurrent thrombosis. Similar upward trends in D-dimer (P=0.001), antithrombin (P=0.001), and prothrombin fragment F1+2 (P=0.001) were observed in the 76 patients who died during the study and in the patients who received chemotherapy. The authors concluded that successful oral anticoagulation with warfarin in patients with cancer and venous thromboembolic disease is more likely to be achieved in patients with early stage tumors and good performance status. The persistence of activation of hemostasis, as shown by plasma coagulation markers, is a strong predictor of recurrence and poor outcome.

Sallah S, Husain A, Sigounas V, et al. Plasma coagulation markers in patients with solid tumors and venous thromboembolic disease receiving oral anticoagulation therapy. Clin Cancer Res. 2004;10:72387243.

Reprints: Sabah Sallah, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71103;

bullet Performance characteristics of automated natriuretic peptide assays

B-type natriuretic peptide is a neurohormone synthesized in the ventricles of the heart as a 134-amino acid polypeptide known as preproBNP, which is cleaved to produce proBNP (108 amino acids) and an N-terminal signal peptide (26 amino acids). ProBNP is cleaved to the inactive N-terminal proBNP (NT-proBNP) fragment (amino acids 176) and the hormone BNP (amino acids 77108). The release of BNP and NT-proBNP is dependent on ventricular myocyte stretch in response to ventricular volume expansion. Measurements of BNP and NT-proBNP have been used to identify patients with heart failure and to monitor the efficacy of their treatment. Human BNP (BNP 132) is biologically active and is composed of the last 32 amino acids of the carboxyl terminus of proBNP. It has a circulating half-life of 20 minutes. Studies have shown that there may be more than one form of circulating BNP. A number of commercially available automated assays for BNP and NT-proBNP measurement are available, including the Access 2 BNP (Biosite), Advia Centaur BNP (Bayer Diagnostics), AxSym BNP (Abbott Diagnostics), and Elecsys NT-proBNP assays (Roche Diagnostics). The authors evaluated these four automated methods for limit of detection, linearity, imprecision, and reference intervals. They also performed method comparison studies in which the Triage point-of-care immunoassay (Biosite) was used as the comparison method, and they assessed analytic concordance. Imprecision testing showed total coefficients of variation of 4.1 percent for the Access 2, 4.4 percent for the Advia Centaur, 5.5 percent for the AxSym, and 0.8 percent for the E170. Relative to the Triage meter, method comparison revealed a slope of 0.96 (r=0.95) for the Access 2, 0.77 (r=0.92) for the Advia Centaur, 1.13 (r=0.94) for the AxSym, and 8.8 (r=0.80) for the E170. Overall analytic concordance values with the Triage meter were 95.9 percent for the Access 2, 92.9 percent for the Advia Centaur, 92.4 percent for the AxSym, and 84.3 percent for the E170. The authors concluded that all automated natriuretic peptide methods showed acceptable analytic performance.

Rawlins ML, Owen WE, Roberts WL. Performance characteristics of four automated natriuretic peptide assays. Am J Clin Pathol. 2005;123:439445.

Reprints: Dr. William L. Roberts, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108

bullet Factors that influence month-of-birth pattern in narcolepsy

Narcolepsy is a debilitating, lifelong disorder characterized by excessive daytime sleepiness and REM sleep abnormalities, including cataplexy, hypnagogic hallucinations, and sleep paralysis. The etiology of human narcolepsy with cataplexy is thought to involve genetic and environmental factors and a possible autoimmune-mediated degeneration of hypocretin (orexin) neurons. Evidence supporting environmental involvement includes the low rate of monozygotic twin concordance and the relative infrequency of familial occurrence. Evidence supporting the involvement of the immune system includes a strong association between narcolepsy with clinically typical or severe cataplexy and HLA genes, especially DQB1*0602. Evidence that environmental factors may play a role during early development was provided in three studies showing unusual birth patterns in narcolepsy patients from the northern hemisphere. Seasonal birth patterns have been found for other CNS disorders, such as Parkinsons disease and schizophrenia, as well as for autoimmune disorders, such as type 1 diabetes mellitus. These patterns have been taken as evidence of exposure to a noxious event during early development. The authors undertook a study to provide additional information about month-of-birth patterns in narcolepsy. The data were obtained from narcolepsy patients taking part in clinical trials assessing the safety and efficacy of modafinil for treating excessive daytime sleepiness. Finding that a month-of-birth effect is independent of genetic susceptibility would raise the possibility of separate etiological mechanisms and provide evidence of disease heterogeneity, with possible diagnostic and treatment implications. The authors conducted a cross-sectional survey using data from clinical trials conducted in sleep clinics throughout the United States and involving 530 narcolepsy patients diagnosed based on the International Classification of Sleep Disorders using clinical histories, nocturnal polysomnography, and Multiple Sleep Latency Tests. A surplus of March births and a fall-off in September births was found in the narcolepsy patients relative to the general population. This finding was observed only when cataplexy was moderate or severe. The month-of-birth pattern was similar for HLA-DQB1*0602-positive and -negative patients. A March birth and HLA-DQB1*0602 positivity were independent risk factors in a logistic regression analysis. The authors concluded that environmental events during development may influence the severity of narcolepsy or the likelihood of developing the disease.

Picchioni D, Mignot EJ, Harsh JR. The month-of-birth pattern in narcolepsy is moderated by cataplexy severity and may be independent of HLA-DQB1*0602. Sleep. 2004;27:14711475.

Reprints: Dr. John Harsh, Box 5025, Dept. of Psychology, University of Southern Mississippi, Hattiesburg, MS 39406-5025;

bullet Urinary excretion of homocysteine-thiolactone

Numerous clinical studies have shown that plasma total homocysteine is a risk factor for cardiovascular disease and stroke and predicts mortality independently of traditional risk factors in patients with coronary artery disease. A possible molecular mechanism underlying homocysteine (Hcy) toxicity involves metabolic conversion of Hcy to Hcy-thiolactone during the steps leading to protein biosynthesis. Because of its similarity to the protein amino acid methionine, Hcy enters the initial steps of protein synthesis and is misactivated by methionyl-tRNA synthetase to form homocysteinyl-adenylate. Hcy-thiolactone can be detrimental because it can modify proteins by forming adducts in which Hcy is N-linked to the e-amino group of protein lysine residues. Modification with Hcy-thiolactone affects protein structure, decreases the physiologic activities of proteins, and has toxic effects on cells. N-Hcy-protein complexes elicit immune responses in rabbits and humans. An autoimmune response to N-Hcy-protein complexes is associated with stroke. Human plasma concentrations of Hcy-thiolactone are much lower than those expected from studies with cultured human cells. For example, in cultured human diploid fibroblasts or umbilical vascular endothelial cells, Hcy-thiolactone represents approximately 10 percent of tHcy. However, Hcy-thiolactone represents less than 0.8 or 0.2 percent of plasma tHcy. This apparent discrepancy suggests that the body possesses mechanisms that prevent Hcy-thiolactone from accumulating to harmful concentrations. The authors used a sensitive high-pressure liquid chromatography method with postcolumn derivatization and fluorescence detection to examine Hcy-thiolactone concentrations in urine and plasma. They discovered a previously unknown pool of Hcy-thiolactone in urine. Urinary concentrations of Hcy-thiolactone (11485 nmol/L; n=19) were approximately 100-fold higher than those in plasma (0.122.6 nmol/L; n=20). Urinary Hcy-thiolactone accounted for 2.5 to 28.3 percent of urinary total Hcy, whereas plasma Hcy-thiolactone accounted for less than 0.002 to 0.29 percent of plasma total Hcy. Urinary concentrations of Hcy-thiolactone, but not of total Hcy, were negatively correlated with urinary pH. Clearance of Hcy-thiolactone, relative to creatinine, was 0.21 to 6.96. In contrast, relative clearance of Hcy was 0.001 to 0.003. The authors concluded that the analytical methods described here can be used to quantify Hcy-thiolactone in biological fluids. Using these methods, the authors showed that the human body eliminates Hcy-thiolactone by urinary excretion. Their data also suggest that the protonation status of its amino group affects Hcy-thiolactone excretion.

Chwatko G, Jakubowski H. Urinary excretion of homocysteine-thiolactone in humans. Clin Chem. 2005;51:408415.

Reprints: Reprints: Hieronym Jakubowski, Dept. of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, NJ 07103;

bullet Correlation between thromboxane A2 and in vitro aggregation in platelets

In vitro tests are used to assess the quality of preserved platelets. Platelet viability has been assessed from pH of the platelet medium, platelet morphology, and platelet response to hypotonic stress. However, data reported on rabbit, mouse, and baboon platelets subjected to degranulation failed to demonstrate a correlation between morphology and the survival of autologous platelets. In these animals, autologous platelets subjected to extensive shape changes had normal in vivo survival values. The function of fresh and preserved platelets is assessed by platelet aggregation response to single and dual agonists. Platelet function can also be assessed by the production of thromboxane A2 after stimulation with agonists while measuring the aggregation response. A decrease in the level of thromboxane A2 in shed blood collected at the bleeding time site was associated with an increase in bleeding time, and an increase in the level of thromboxane A2 in shed blood was associated with a decrease in bleeding time. In the authors in vitro study of human fresh, liquid-preserved, and cryopreserved platelets, they measured the response to aggregation and the production of thromboxane A2 after stimulation. They evaluated the effects of single and dual agonists, concentration of agonists, pH, and plasma and nonplasma resuspension media on platelet aggregation and thromboxane A2 production. Platelets isolated by apheresis procedures were stored at 220C for up to five days and then frozen with six percent dimethyl sulfoxide, stored at 800C, thawed, washed, and resuspended in medium. The authors measured the effects of agonists and pH and composition of the medium on platelet aggregation and platelet production of thromboxane A2 after stimulation. The agonists and the pH and composition of the medium affected the aggregation response and production of thromboxane A2 by the fresh and preserved platelets. Platelet aggregation response to arachidonic acid and adenosine diphosphate was significantly lower in the cryopreserved platelets than in the fresh and preserved platelets. After stimulation with arachidonic acid and adenosine diphosphate, the cryopreserved platelets produced more thromboxane than did the fresh and liquid-preserved platelets. The authors concluded that the agonists and the pH and composition of the medium affected the response to aggregate and produced thromboxane in vitro in fresh and liquid-preserved platelets. Platelet thromboxane A2 production may be a better in vitro test than platelet aggregation to assess platelet function in vivo.

Valeri CR, Macgregor H, Ragno G. Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: effects of agonists, pH, and plasma and saline resuspension. Transfusion. 2005;45:596603.

Reprints: Reprints: Dr. C. Robert Valeri, Naval Blood Research Laboratory, Boston University School of Medicine, 615 Albany St., Boston, MA 02118;


Dr. Cibull is professor of pathology and laboratory medicine and direct of surgical pathology, University of Kentucky Medical Center, Lexington. Dr. Lele is assistant professor of pathology and laboratory medicine, University of Kentucky Medical Center. Dr. Kesler is hematopathology fellow, University of Texas Southwestern Medical Center at Dallas.