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Further work is needed to determine whether changes in fsTnI and/or ssTnI are specific for a given disease (and if so, its severity) and particular muscle types. In addition, the performance of WB-DSA, like any other diagnostic assay using antibodies, is limited by antibody selection and the possibility that modifications of the target protein alter binding affinities and, hence, assay results. It will therefore be necessary to screen different patient cohorts with a variety of antibodies to overcome such limitations. Nevertheless, preferential or selective release of the two isoforms (and their modified products) into blood raises the possibility of improving the differential diagnosis of skeletal muscle injuries or disease, prognosis, and the evaluation of therapeutic effectiveness.
This work was supported by grants to S. I. and J. V. E. from the Canadian Institutes of Health Research (MOP 36339 and MT 14375), the Heart and Stroke Foundation of Canada (T-3759), and the Ontario Thoracic Society. We thank Spectral Diagnostics Inc. (Toronto, Canada) for generously providing one of the antibodies for this study. References 1. Joint European Society of Cardiology/American College of Cardiology Committee. Myocardial infarction redefined: a consensus document of the joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. J Am Coll Cardiol 2000;36:954 – 69. 2. Braunwald E, Antman EM, Beasley JW, Califf RM, Cheitin MD, Hochman JS, et al. ACC/AHA guidelines for the management of patients with unstable angina and non-ST-segment elevation myocardial infarction. J Am Coll Cardiol 2000;36:970 –1062. 3. Gunst JJ, Langlois MR, Delanghe JR, De Buyzere ML, Leroux-Roels GG. Serum creatine kinase activity is not a reliable marker for muscle damage in conditions associated with low extracellular glutathione concentration. Clin Chem 1998;44:939 – 43. 4. Rama D, Margaritis I, Orsetti A, Marconnet P, Gros P, Larue C, et al. Troponin I immunoenzymometric assays for detection of muscle damage applied to monitoring a triathlon. Clin Chem 1996;42:2033–5. 5. Sorichter S, Mair J, Koller A, Gebert W, Rama D, Calzolari C, et al. Skeletal troponin I as a marker of exercise-induced muscle damage. J Appl Physiol 1997;83:1076 – 82. 6. Sorichter S, Mair J, Koller A, Calzolari C, Huonker M, Pau B, et al. Release of muscle proteins after downhill running in male and female subjects. Scand J Med Sci Sports 2001;11:28 –32. 7. Onuoha GN, Alpar EK, Dean B, Tidman J, Rama D, Laprade M, et al. Skeletal troponin-I release in orthopedic and soft tissue injuries. J Orthop Sci 2001;6:11–5. 8. Labugger R, Organ L, Collier C, Atar D, Van Eyk JE. Extensive troponin I and T modification detected in serum from patients with acute myocardial infarction. Circulation 2000;102:1221– 6. 9. Simpson JA, Van Eyk JE, Iscoe S. Hypoxemia-induced modification of troponin I and T in canine diaphragm. J Appl Physiol 2000;88:753– 60. 10. Ariano MA, Armstrong RB, Edgerton VR. Hindlimb muscle fiber populations of five mammals. J Histochem Cytochem 1973;21:51–5. 11. Matsumoto N, Nakamura T, Yasui Y, Torii J. Immunohistochemical differentiation of fiber types in human skeletal muscle using monoclonal antibodies to slow and fast isoforms of troponin I subunit. Biotech Histochem 1997; 72:191–7. 12. Wilkinson JM, Grand RJ. Comparison of amino acid sequence of troponin I from different striated muscles. Nature 1978;271:31–5. 13. Lavoinne A, Hue G. Serum cardiac troponins I and T in early posttraumatic rhabdomyolysis. Clin Chem 1998;44:667– 8. 14. Stelow EB, Johar VP, Smith SA, Crosson JT, Apple FS. Propofol-associated rhabdomyolysis with cardiac involvement in adults: chemical and anatomic findings. Clin Chem 2000;46:577– 81. 15. Benoist J, Cossen C, Mimoz O, Edouard A. Serum cardiac troponin I, creatine kinase (CK), and CK-MB in early posttraumatic rhabdomyolysis [Letter]. Clin Chem 1997;43:416 –7. 16. Lofberg M, Tahtela R, Harkonen M, Somer H. Myosin heavy-chain fragments
and cardiac troponins in the serum in rhabdomyolysis. Diagnostic specificity of new biochemical markers. Arch Neurol 1995;52:1210 – 4. 17. Omar MA, Wilson JP, Cox TS. Rhabdomyolysis and HMG-CoA reductase inhibitors. Ann Pharmacother 2001;35:1096 –107.
Preanalytical Influences on the Measurement of Ghrelin, Michael Gro¨ schl,* Roland Wagner, Jo¨ rg Do¨ tsch, Wolfgang Rascher, and Manfred Rauh (Kinderklinik Erlangen, Loschgestrasse 15, 91054 Erlangen, Germany; * author for correspondence: fax 0049-09131-8533745, e-mail michael.
[email protected]) Ghrelin is an acylated peptide with growth-hormonereleasing function (1, 2 ). It was first isolated from rat stomach during the search for an endogenous ligand to an “orphan” G-protein-coupled receptor (3 ). The peptide consists of 28 amino acids, with a n-octanoylation of the serine-3 residue, which is indispensable for biological activity. Human ghrelin differs from rat ghrelin by only two amino acids at positions 11 and 12. The peptide stimulates the release of growth hormone when administered intravenously to rats and given to rat primary pituitary cells (2 ). In previous studies, serum was preferred for the determination of ghrelin. Experience with other sample materials obtained after administration of various anticoagulating substances has not yet been described. It is therefore unknown which method of obtaining samples for ghrelin determination enables the most accurate and precise measurements. Furthermore, data on the stability of the hormone are still lacking, but are necessary for optimizing analytical conditions. The objective of the present study was to compare the reliability of ghrelin measurements in serum and four different plasma samples and to evaluate data on stability under different storage conditions. Blood samples were taken from apparently healthy volunteers (10 men and 4 women; age range, 18 – 40 years) who were not on medication and had normal blood pressure. The body mass index varied from 20 to 29 kg/m2. Blood was taken between 1000 and 1100 by venipuncture (Multifly® with 20-mL cannulas; Sarstedt) and immediately divided into tubes for plasma preparation with dipotassium EDTA (Kabe), citrate, fluoride, and lithium heparinate (Sarstedt) as anticoagulating substances. The content of liquid anticoagulating additive in citrate-plasma tubes was 118 ⫾ 15 L (n ⫽ 15; mean ⫾ SD). Additionally, serum was prepared from each sample (Sarstedt). After clotting, samples were centrifuged (10 min at 1500g). Serum from five male volunteers was divided into two series of five aliquots each. One of the duplicate series was stored at 25 °C, the other was stored at 4 °C. Each day an additional sample from each series was frozen (⫺25 °C) until measurement. To study the effect of repeated freezing and thawing, sera from healthy volunteers (n ⫽ 10) were divided into
Clinical Chemistry 48, No. 7, 2002
five identical aliquots. All aliquots were frozen immediately; four of these were rethawed the next day and then refrozen, with three then being rethawed, and so forth. Additionally, blood from one male volunteer was supplemented with 150 or 500 ng/L recombinant human ghrelin. The blood was divided between diverse matrices as described above, and each sample was measured 10 times. Recovery of the added amounts was determined after subtraction of the basal ghrelin value of the sample. Ghrelin was measured with a commercial RIA (Phoenix). Fifty percent binding occurred at 190 ng/L. The sensitivity of undiluted samples was 15 ng/L. Inter- and intraassay CVs, as given by the manufacturer, were 7.5% and 4.0%, respectively. Values in different matrices were compared by Passing–Bablok regression (4 ). The CV for any sample matrix was the mean intraassay CV from all of the different samples. ANOVA with the Bonferroni multiple comparison test was used to examine alterations in hormone values under various storage conditions and to assess the influences of repeated freezing and thawing. Alterations exceeding ⫾ 2 intraassay CVs were defined as being stability dependent. P ⬍0.05 was considered significant. The mean intraassay CV of ghrelin measurements from serum was 3.8%. Because the determination of ghrelin from serum is commonly used, all further descriptions of different matrices are related to the ghrelin measurements in this matrix. As in serum, we found a low intraassay CV of 3.8% for ghrelin measurements in dipotassium-EDTA plasma. The linear regression equation between both matrices was as follows: dipotassium-EDTA plasma ⫽ 1.01 ⫻ serum ⫹ 12.3 ng/L. The differences from the respective serum values were ⫺33% to ⫹14% with a correlation of r2 ⫽ 0.97. There was no significant difference between ghrelin concentrations from matched serum and EDTA-plasma samples. The intraassay CV of ghrelin measurements from lithium-heparinate plasma was 4.8%. The differences from the corresponding serum values were ⫺29% to ⫹39% with a correlation of r2 ⫽ 0.95, and the regression equation was as follows: lithium-heparinate plasma ⫽ 1.07 ⫻ serum ⫺ 17.8 ng/L. This matrix generally yielded significantly lower results (mean, 7%; P ⬍0.01) compared with the matched serum samples. In fluoride-plasma tubes, the intraassay CV of ghrelin measurements was 4.5%. Here, the differences from the matched serum values were ⫺30% to ⫹15% with a correlation of r2 ⫽ 0.90. The regression equation was as follows: fluoride plasma ⫽ 0.84 ⫻ serum ⫹ 14.4 ng/L. Significant differences in comparison with serum values were not found. Significantly lower concentrations of ghrelin were measured in citrate plasma in comparison with serum (P ⬍0.001). The results were generally 25% lower than in the serum samples with a range of ⫺51% to ⫹2%. The correlation to the matched serum samples was r2 ⫽ 0.94. The CV was 2.8%. The regression equation was as follows: citrate plasma ⫽ 0.71 ⫻ serum ⫹13 ng/L.
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After subtraction of the basal ghrelin content measured in each of the five sample matrices, we calculated recoveries (%) of the added amounts of recombinant ghrelin. Recovery of 150 ng/L recombinant ghrelin (mean ⫾ SD) was 100% ⫾ 13% in serum, 99% ⫾ 12% in EDTA plasma, 94% ⫾ 4% in lithium-heparinate plasma, 100% ⫾ 11% in fluoride plasma, and 83% ⫾ 10% in citrate plasma. The recoveries for 500 ng/L ghrelin added to various matrices were 101% ⫾ 5% in serum, 98% ⫾ 6% in EDTA plasma, 100% ⫾ 7% in lithium-heparinate plasma, 97% ⫾ 7% in fluoride plasma, and 88% ⫾ 6% in citrate plasma. Ghrelin was stable when stored at 4 °C for up to 3 days, whereas storage at 25 °C for ⬎1 day produced significantly lower results (Fig. 1). Repeated freezing and thawing had no influence on the concentrations of the peptide (P ⫽ 0.39). Since its discovery in 1999, many studies on ghrelin have been published. Most studies describe the use of serum (5, 6 ) whereas only a few used plasma, and these studies had no further explanation regarding plasma use (7, 8 ). Because of the increased interest in measuring ghrelin, a standardized method for sample collection is required. Consequently, our aim was to compare the equivalence of ghrelin values measured from different specimens and to determine the optimal sample matrix for accuracy and reliability, as we recently described for human leptin (9 ). Our additional aim was to investigate the stability of ghrelin under various storage conditions because previous studies have described the influence of storage conditions on the analysis of various endocrine substances, such as steroids (10, 11 ) and peptide hormones, including human growth hormone (12 ), lutropin, follitropin, and prolactin (13 ). Our results show only slight differences in the ghrelin measurements in serum and different plasmas obtained from identical blood samples. Data on the intraassay CV for serum were in good accordance with data provided by the manufacturer. All five matrices showed low intraas-
Fig. 1. Stability of human ghrelin after storage at 25 and 4 °C for up to 5 days (mean ⫾ SD). Dotted lines indicate clinical acceptable range (⫾ 3 intraassay CVs).
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say CVs. The high precision of the results may enable single measurements instead of multiple determination, thereby reducing costs. The significantly lower values for ghrelin in citrate plasma may only partially (⬃12%) be explained by dilution with the anticoagulating liquid in the tubes (118 ⫾ 15 L). The discrepancy between citrate plasma and serum was ⬃25%, a discrepancy that is too high for a recommendation for citrate plasma. In contrast, results for lithiumheparinate plasma were in only ⬃7% lower than serum results, which we consider to acceptable. It should be kept in mind that the magnitude of the difference between values from matched sample matrices might be influenced by the assay system used, as has been shown for the determination of cardiac troponin T and I (14, 15 ). Our findings are based on the use of a direct RIA that is commercially available and is currently widely used for research. As we have determined, storage of serum under cooled conditions allows stable results for up to 3 days. Storage at warm temperatures for ⬎1 day should be avoided. This is very important when samples are transported by mail. Because no significant decrease in the ghrelin values was observed after repeated freezing and thawing, there should be no problems if sample tubes are used several times, e.g., for repeating an assay or using material after determination of other analytes. In conclusion, ghrelin is relatively stable when stored under cooled conditions. This, as well as the fact that several sample matrices can be used as alternatives, is a good precondition for further studies on this interesting peptide hormone. References 1. Date Y, Murakami N, Kojima M, Kuroiwa T, Matsukura S, Kangawa K, et al. Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats. Biochem Biophys Res Commun 2000;275:477– 80. 2. Dieguez C, Casanueva FF. Ghrelin: a step forward in the understanding of somatotroph cell function and growth regulation. Eur J Endocrinol 2000;142: 413–7. 3. Kojima M, Hosoda H, Matsuo H, Kangawa K. Ghrelin: discovery of the natural endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol Metab 2001;12:118 –22. 4. Bablok W, Passing H. Application of statistical procedures in analytical instrument testing. J Autom Chem 1985;7:74 –9. 5. Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, et al. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001;86: 5083– 6. 6. Caixas A, Bashore C, Nash W, Pi-Sunyer F, Laferrere B. Insulin, unlike food intake, does not suppress ghrelin in human subjects. J Clin Endocrinol Metab 2002;87:1902. 7. Makino Y, Hosoda H, Shibata K, Makino I, Kojima M, Kangawa K, et al. Alteration of plasma ghrelin levels associated with the blood pressure in pregnancy. Hypertension 2002;39:781– 4. 8. Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M, et al. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 2002;87:240 – 4. 9. Gro¨ schl M, Wagner R, Do¨ rr HG, Blum WF, Rascher W, Do¨ tsch J. Variability of leptin values measured from different sample matrices. Horm Res 2000; 54:26 –31. 10. Gro¨ schl M, Wagner R, Rauh M, Do¨ rr HG. Stability of salivary steroids: the influences of storage, food and dental care. Steroids 2001;66:737– 41. 11. Dabbs JMJ. Salivary testosterone measurements: collecting, storing, and mailing saliva samples. Physiol Behav 1991;49:815–7. 12. Dattani MT, Ealey PA, Pringle PJ, Hindmarsh PC, Brook CG, Marshall NJ. An investigation into the lability of the bioactivity of human growth hormone using the ESTA bioassay. Horm Res 1996;46:64 –73.
13. Kubasik NP, Ricotta M, Hunter T, Sine HE. Effect of duration and temperature of storage on serum analyte stability: examination of 14 selected radioimmunoassay procedures. Clin Chem 1982;28:164 –5. 14. Stiegler H, Fischer Y, Vazquez-Jimenez JF, Graf J, Filzmaier K, Fausten B, et al. Lower cardiac troponin T and I results in heparin-plasma than in serum. Clin Chem 2000;46:1338 – 44. 15. Gerhardt W, Nordin G, Herbert AK, Burzell BL, Isaksson A, Gustavsson E, et al. Troponin T and I assays show decreased concentrations in heparin plasma compared with serum: lower recoveries in early than in late phases of myocardial injury. Clin Chem 2000;46:817–21.
Biological Variation of Glycohemoglobin, Curt Rohlfing,1* Hsiao-Mei Wiedmeyer,1 Randie Little,1 V. Lee Grotz,2 Alethea Tennill,1 Jack England,1 Richard Madsen,1 and David Goldstein1 (1 University of Missouri School of Medicine, Columbia, MO 65212; 2 McNeil Specialty Products Company, New Brunswick, NJ 08903; * address correspondence to this author at: Department of Child Health, University of Missouri–Columbia, 1 Hospital Dr., M772, Columbia, MO 65212; fax 573-884-4748, e-mail RohlfingC@ health.missouri.edu) Glycohemoglobin (GHb) is a measure of long-term mean glycemia that predicts risks for the development and/or progression of diabetic complications in patients with type 1 and type 2 diabetes (1, 2 ). Several reports have suggested, however, that although the within-subject variation in GHb unrelated to glycemia is minimal, there is substantial between-subject variation in GHb, e.g., “low glycators” and “high glycators” (3–5 ). These reports have suggested that because of this large between-subject variation, GHb may not be useful for diabetes screening or diagnosis and that when GHb is used for routine management of patients with diabetes, different patients may require very different GHb target values to achieve the same overall glycemic status. We therefore examined the biological variation of GHb and fasting plasma glucose (FPG) in nondiabetic individuals. Individuals without diabetes (n ⫽ 48) participated in a study of an artificial sweetener that has no effect on GHb or plasma glucose concentrations [Submission to Food and Drug Administration. McNeil Specialty Products Company food additive petition 7A3987 (Sucralose), 1987–1997]. Because the study was designed to detect minimal changes in plasma glucose concentrations, all participants were men to avoid the effects of cyclic hormonal changes on insulin (and therefore, plasma glucose) concentrations. At the prestudy screening, all individuals were healthy on the basis of a medical history, physical examination, and electrocardiography results; results of hematology and blood chemistry studies, urine examination, and measures of blood glucose control (FPG, insulin, C-peptide, and hemoglobin A1c) were all within their respective reference intervals. Participants who failed a baseline oral glucose tolerance test [fasting ⬎7.8 mmol/L (140 mg/dL), 1 h ⬎11.1 mmol/L (200 mg/dL), and/or 2 h ⬎7.8 mmol/L (140 mg/dL)] were excluded. Those who took medications that could affect glucose
