Clinical Abstracts

June 2005

Editors:
Michael Bissell, MD, PhD, MPH,
Ronald Domen, MD,

Utility of serum free cortisol
Efficacy of testing kits for the drug MDMA
Glucose-6-phosphate isomerase autoantibodies in rheumatoid arthritis
Cancers of multiple origins show PML loss
Link between oxidative stress and type 2 diabetes

Utility of serum free cortisol

Patients with critical illnesses have elevated glucocorticoid secretion marked by an increase in the serum total cortisol concentration. Assays for serum cortisol measure the total hormone concentration (serum free cortisol plus the protein-bound fraction of cortisol). The current consensus is that free cortisol, rather than the protein-bound fraction, is responsible for the physiologic function of the hormone. Because more than 90 percent of circulating cortisol in human serum is bound to proteins (corticosteroid-binding globulin and albumin), it is reasonable to suggest that alterations in the binding proteins could affect measured concentrations of serum total cortisol and, thus, the interpretation of tests used to assess adrenal function. In critical illness, measured serum total cortisol concentrations can be misleadingly lower than anticipated, resulting in the incorrect conclusion that adrenal function is impaired. The authors evaluated adrenal function by measuring serum total cortisol and free cortisol concentrations in critically ill patients without known adrenal dysfunction. Baseline serum total cortisol, cosyntropin-stimulated serum total cortisol, aldosterone, and free cortisol concentrations were measured in 66 critically ill patients and 33 healthy volunteers in groups that were similar with regard to gender and age. Of the 66 patients, 36 had hypoproteinemia (albumin concentration, 2.5 g/dL or less) and 30 had near normal serum albumin concentrations (above 2.5 g/dL). Baseline and cosyntropin-stimulated serum total cortisol concentrations were lower in the patients with hypoproteinemia than in those with near-normal serum albumin concentrations (P<0.001). However, the mean baseline serum free cortisol concentrations were similar in the two groups of patients (5.1±4.2 and 5.2±3.5 µg/dL [140.7±113.1 and 143.5±96.6 nmol/L]) and were several times higher than the values in controls (0.6±0.3 µg/dL [16.6±8.3 nmol/L], P<0.001 for both comparisons). Cosyntropin-stimulated serum total cortisol concentrations were subnormal (18.5 µg/dL [510.4 nmol/L] or less) in 14 of the patients, all of whom had hypoproteinemia. In all 66 patients, including the 14 who had hypoproteinemia, the baseline and cosyntropin-stimulated serum free cortisol concentrations were high-normal or elevated. The authors concluded that during critical illness, glucocorticoid secretion markedly increases, but the increase is not discernible when only the serum total cortisol concentration is measured. In this study, nearly 40 percent of critically ill patients with hypoproteinemia had subnormal serum total cortisol concentrations even though their adrenal function was normal. Measuring serum free cortisol concentrations in critically ill patients with hypoproteinemia may help prevent the unnecessary use of glucocorticoid therapy.

Hamrahian AH, Oseni TS, Arafah BM. Measurements of serum free cortisol in critically ill patients. N Engl J Med. 2004; 350: 1629–1638.

Reprints: Dr. B.M. Arafah, Division of Clinical and Molecular Endocrinology, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106; bxa@po.cwru.edu

Efficacy of testing kits for the drug MDMA

Use of the club drug 3,4-methylenedioxymethamphetamine (MDMA), known as Ecstasy, has risen in recent years. Although MDMA is a dangerous drug, additional problems arise from contaminants found in street samples and from counterfeit tablets in which other ingredients have been substituted for MDMA. Because these drugs are produced in clandestine laboratories, they are seldom pure, and the amount of drug in a capsule or tablet is likely to vary considerably from one source to another. Three drugs known to be mixed with or substituted for MDMA are paramethoxyamphetamine (PMA, a stimulant similar in effect to MDMA), dextromethorphan (a cough suppressant with dissociative effects in high doses), and methamphetamine (known as speed, crystal meth, or crank). Organizations loosely referred to as harm-reduction groups promote the public use of colorimetric test kits for differentiating between authentic MDMA and contaminated or adulterated products. Many dangers are associated with field drug testing. For instance, most test users are young and inexperienced and may perform the tests under the intoxicating effects of ethanol or other drugs. In addition, these tests may lack the precision (reproducibility) and accuracy (correct identification) necessary to positively predict the purity of MDMA tablets. Further, since users regard MDMA as a benign drug, the temptation will be to assume the pill being evaluated is MDMA even though the subjective test results may be questionable. The authors evaluated the DanceSafe complete adulterant screening kit for its accuracy in identifying MDMA and methylenedioxyamphetamine (MDA) derivatives and for its ability to detect certain contaminants. Thirty-nine street-grade tablets purported to be MDMA were tested with the Marquis, Mecke, and Simon’s reagents provided in the DanceSafe testing kit. The tablets were then submitted for gas chromatography-mass spectrometry to identify the active ingredients. The seven known drugs-of-abuse—codeine, dextromethorphan, dihydrocodeine, ketamine, MDMA, morphine, and d-norpropoxyphene—were tested with Marquis, Mecke, and Simon’s reagents. The reagents did not differentiate pure MDMA from adulterated forms. They lacked sensitivity and specificity for identifying MDMA when used by people unfamiliar with these reagents. Furthermore, experienced toxicologists using this unfamiliar procedure generated false-positive results. The authors concluded that neither Marquis, Mecke, nor Simon’s reagents should be used by the public for harm-reduction purposes. These reagents do not help identify pure MDMA tablets. Gas chromatography-mass spectrometry remains the most sensitive and specific test for identifying MDMA and its contaminants.

Murray RA, Doering PL, Boothby LA, et al. Putting an Ecstasy test kit to the test: harm reduction or harm induction? Pharmacotherapy. 2003;23:1238–1244.

Reprints: Dr. Lisa A. Boothby, BCPS, 710 Center St., Columbus, GA 31902; lisa.boothby@crhs.net

Glucose-6-phosphate isomerase autoantibodies in rheumatoid arthritis

One feature of rheumatoid arthritis is the presence of antibodies against self-antigens inside and outside the joint. Detection of autoantibodies such as rheumatoid factors is commonly used as a diagnostic and prognostic tool. It has been shown in animal models that autoantibodies against glucose-6-phosphate isomerase (GPI) are present in high titers and cause disease on transfer into healthy mice. Because of the dominant role of GPI antibodies in this animal model, antibody titers against GPI were studied in humans. It has been reported that GPI is an autoantigen in rheumatoid arthritis, being present in 64 percent of patients but only a few healthy control subjects. However, this assertion is surrounded by controversy since a number of other groups of investigators could not reproduce these observations. Patients with rheumatoid arthritis are heterogeneous in their autoantibody response. While approximately 70 to 80 percent of rheumatoid arthritis patients have rheumatoid factors, other autoantibodies, such as anti-C1q antibodies and antineutrophil cytoplasmic antibodies, are less common and are found in patients with more severe disease. Therefore, the apparently conflicting findings of anti-GPI antibodies in rheumatoid arthritis may be explained by their occurrence at a specific point in the disease process. The authors studied groups of rheumatoid arthritis patients at different stages of disease to determine whether autoantibodies to GPI occur in rheumatoid arthritis, and if so, at what stage of the disease. Using an enzyme-linked immunosorbent assay, serum from 131 rheumatoid arthritis patients and 28 healthy controls was tested for autoantibodies against recombinant human GPI. Patients were grouped according to disease duration and presence of rheumatoid nodules, rheumatoid vasculitis, and Felty’s syndrome, which are extra-articular complications of the disease. Elevated levels of autoantibodies against GPI were present in five percent of patients with uncomplicated rheumatoid arthritis and four percent of controls. In rheumatoid arthritis complicated by extra-articular manifestations, anti-GPI antibodies were observed in 18 percent of patients with rheumatoid nodules, 45 percent of patients with rheumatoid vasculitis, and 92 percent of patients with Felty’s syndrome. The authors concluded that in rheumatoid arthritis patients, autoantibodies to GPI are associated with the occurrence of extra-articular complications.

Van Gaalen FA, Toes REM, Ditzel HJ, et al. Association of autoantibodies to glucose-6-phosphate isomerase with extraarticular complications in rheumatoid arthritis. Arthritis Rheum. 2004;10:395–399.

Reprints: Dr. Floris A. van Gaalen, Dept. of Rheumatology, Leiden University Medical Center, P.O. Box 9600 2300 RD, Leiden, Netherlands; f.a.van_gaalen@lumn.nl

Cancers of multiple origins show PML loss

In acute promyelocytic leukemia, the PML gene (on chromosome 15) is frequently fused to the retinoic acid receptor alpha (RARa) gene (on chromosome 17) as a consequence of reciprocal and balanced chromosomal translocation resulting in the production of a PML-RARa fusion protein. PML and the PML nuclear body are essential for critical tumor-suppressive pathways. PML acts as a p53 transcriptional coactivator and is required for p53-dependent induction of apoptosis and cellular senescence. The role of PML in the pathogenesis of human cancers other than acute promyelocytic leukemia (APL) is still unclear. The authors, therefore, assessed the status and expression of the PML gene in solid tumors of multiple histologic origins. They created tumor tissue microarrays using samples from patients with colon adenocarcinoma (n=109), lung carcinoma (n=19), prostate adenocarcinoma (n=36), breast carcinoma (n=38), central nervous system tumors (n=51), germ cell tumors (n=60), thyroid carcinoma (n=32), adrenal cortical carcinoma (n=12), and non-Hodgkin’s lymphoma (n=251), as well as from normal tissue corresponding to each histotype. The authors then analyzed PML protein and mRNA expression by immunohistochemistry and in situ hybridization, respectively. DNA from microdissected tumor samples and cell lines were analyzed for PML mutations and loss of heterozygosity. For some tumor types, the association between PML expression and tumor stage and grade was analyzed. All normal tissues expressed PML protein. PML protein expression was reduced or abolished in prostate adenocarcinomas, colon adenocarcinomas, breast carcinomas, lung carcinomas, lymphomas, tumors of the central nervous system, and germ cell tumors, but not in thyroid or adrenal carcinomas. Loss of PML protein expression was associated with tumor progression in prostate cancer (the progression from prostatic intraepithelial neoplasia to invasive carcinoma was associated with complete PML loss [P<.001]), breast cancer (complete PML loss was associated with lymph node metastasis [P=.01]), and tumors of the central nervous system (complete PML loss was associated with high-grade tumors [P=.003]). PML mRNA was expressed in all tumor and cell line samples. The PML gene was rarely mutated and was not subject to loss of heterozygosity. The authors concluded that PML protein expression is frequently lost in human cancers of various histologic origins and that its loss associates with tumor grade and progression in some tumor histotypes.

Gurrieri C, Capodieci P, Bernardi R, et al. Loss of the tumor suppressor PML in human cancers of multiple histologic origins. J Natl Cancer Inst. 2004;96:269–279.

Reprints: Dr. Pier Paolo Pandolfi, Molecular Biology Program and Dept. of Pathology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Box 110, 1275 York Ave., New York, NY 10021; ppandolfi@ski.mmskcc.org

Link between oxidative stress and type 2 diabetes

Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic ß-cell failure and is associated with the development of atherosclerosis. Atherosclerosis is considered to be, in part, a consequence of chronic low-grade inflammation, and it has been suggested that atherosclerosis and diabetes might share the same inflammatory basis. Oxidative stress is suggested to be associated with macrovascular and microvascular diabetes complications and could thus be another possible pathogenic link between atherosclerosis and diabetes. Prostaglandin-like F₂-isoprostanes are formed during free radical-catalyzed, nonenzymatic peroxidation of arachidonic acid and are reliable indicators of oxidative stress in vivo. One major F₂-isoprostane, 8-iso-PGF₂, is associated with several risk factors for atherosclerosis, including diabetes, smoking, and hypercholesterolemia. The authors conducted a study to make a cross-sectional investigation of the association between type 2 diabetes and a marker of COX-mediated inflammation (15-keto-dihydro-PGF₂), cytokine-mediated inflammation (CRP, serum amyloid protein A [SAA]), and markers of oxidative stress (8-iso-PGF₂, a-tocopherol) in a population-based study. A secondary aim was to study diabetes duration and COX-mediated inflammation and oxidative stress. The authors measured 15-keto-dihydro-PGF₂(a metabolite of prostaglandin F₂), CRP, SAA, 8-iso-PGF₂ (a nonenzymatic, free radical product of arachidonic acid), and a-tocopherol in a population-based sample of 77-year-old men (n=765), in which 112 men had type 2 diabetes. The inflammatory indicators were increased in men with diabetes (urinary 154-keto-dihydro-PGF₂, P<0.001; CRP and SAA, P<0.05). However, when adjusted for body mass index, waist circumference, or fasting insulin, no association was found between diabetes and CRP or SAA. The oxidative stress indicator 8-iso-PGF₂ in urine was increased (P<0.01) in men with diabetes. Patients who were newly diagnosed with diabetes (less than seven years since diagnosis) had increased urinary 15-keto-dihydro-PGF₂ and decreased alpha-tocopherol, but 8-iso-PGF₂ was unaltered. The authors claim this is the first study to show that type 2 diabetes in elderly men is related to COX-mediated inflammation, reflected by enhanced prostaglandin formation. The high levels of cytokine-mediated acute-phase proteins observed in men with diabetes appear to be related to obesity and increased fasting insulin. The results further suggest that the appearance of chronic inflammation is an early process in the pathogenesis of diabetes, whereas oxidative injury may be a later process, possibly related to inflammation.

Helmersson J, Vessby B, Larson A, et al. Association of type 2 diabetes with cyclooxygenase-mediated inflammation and oxidative stress in an elderly population. Circulation. 2004;109:1729–1734.

Reprints: Dr. Samar Basu, Sections of Geriatrics and Clinical Nutrition Research,
Uppsala University, Box 609, SE-751 25 Uppsala, Sweden; samar.basu@pubcare.uu.se