Clinical Chemistry 50, No. 8, 2004
Determination of Guanidinoacetate and Creatine in Urine and Plasma by Liquid Chromatography–Tandem Mass Spectrometry, Se´bastien Cognat,1* David Cheillan,1 Monique Piraud,1 Birthe Roos,2 Cornelis Jakobs,2 and Christine Vianey-Saban1 (1 Service de Biochimie Pe´ diatrique, Hoˆ pital Debrousse, Lyon, France; 2 Department of Clinical Chemistry and Pediatrics, VU University Medical Center, Amsterdam, The Netherlands; * address correspondence to this author at: Service de Biochimie Pe´ diatrique, Hoˆ pital Debrousse, 29 rue Sœur Bouvier, 69322 Lyon cedex 05, France; fax 33-4-7238-5884, e-mail [email protected]
chu-lyon.fr) In the last 10 years, three new inborn errors of creatine metabolism and transport, called creatine deficiency syndromes (CDS), have been described (1 ). These are deficiencies of arginine:glycine amidinotransferase (EC 18.104.22.168), S-adenosyl-l-methionine:guanidinoacetate N-methyltransferase (GAMT; EC 22.214.171.124), and creatine transporter. Patients with CDS have mental retardation, severe speech disturbance, and depending on the disorder, epilepsy and/or extrapyramidal signs. Biochemical detection of CDS relies on the determination of two main metabolites in biological fluids: guanidinoacetate (GAA) and creatine. Few patients with CDS have been reported to date, probably because of underdiagnosis, most likely attributable to the limited availability of quantitative GAA and creatine assays. This underdiagnosis is particularly harmful because some patients improve with oral creatine supplementation. Several analytical methods for GAA and creatine have been described, including use of the Sakaguchi reaction (2 ), stable-isotope-dilution gas chromatography–mass spectrometry (GC/MS) (3, 4 ), and HPLC (5 ). Recently, a liquid chromatography–tandem mass spectrometry (LC/ MS/MS) method has been reported for analysis of these metabolites in plasma and dried blood on filter cards (6 ). Here we describe the validation of LC/MS/MS for the simultaneous quantification of GAA and creatine in urine and plasma and compare the method with a stableisotope-dilution GC/MS method. To investigate the molecular fragmentation and to prepare calibrators, we prepared 5 mmol/L stock solutions and successive dilutions of creatine, GAA (SigmaAldrich), N-methyl-d3-creatine (CDN Isotopes), and [13C2]GAA (Dr. H. Ten Brink, VU University Medical Center, Amsterdam, The Netherlands) in distilled water. We evaluated MS/MS conditions in the positive-ion mode by infusion of aqueous solutions into the API 2000 tandem mass spectrometer (Sciex Applied Biosystems) as described previously (7 ). Nitrogen was used as both the curtain and collision gas. The transitions chosen for the quantification of GAA, creatine, d3-creatine, and [13C2]GAA (multiple-reaction monitoring mode) were m/z 1743101 [declustering potential (DP), 15V; collision energy (CE), 18V]; 188390 (DP, 20V; CE, 26V); 191393 (DP, 20V; CE, 26V); and 1763103 (DP, 15V; CE, 20V), respectively. We mixed 50 L of samples (0, 50, and 500 mol/L
aqueous calibrators; plasma; and 10-fold-diluted urines) with 50 L of water, 30 L of 50 mol/L d3-creatine, and 30 L of 50 mol/L [13C2]GAA. We added 400 L of ethanol (Carlo-Erba) and 400 L of n-hexane (Merck) to each sample. The vials were then mixed for 2 min. The hexane was removed, and vials were centrifuged for 5 min at 17 530g. The supernatants were transferred to new vials and evaporated to dryness under nitrogen at room temperature. The remaining residues were subsequently derivatized with 200 L of 3 mol/L HCl in n-butanol (Regis Technologies Inc.) for 15 min at 65 °C. Samples were dried again under nitrogen at 45 °C, and 500 L of mobile phase was added. We injected 5 L in the LC/ MS/MS system. Liquid chromatography was performed with a 2.1 ⫻ 50 mm Symmetry C18 HPLC column (3.5-m particle size; Waters). The column flow rate was 0.150 mL/min, and the mobile phase consisted of acetonitrile (200 mL/L in water) acidified with formic acid (0.5 mL/L). A Series 200 micropump and autosampler (Perkin-Elmer) were used for solvent delivery and sample introduction. The elution time was 3 min. The eluate was injected in the TurboIonSpray probe (350 °C) of the mass spectrometer, and results were acquired with Analyst (Ver. 1.3.1) software (Sciex Applied Biosystems). GAA and creatine were quantified relative to the [13C2]GAA and the d3-creatine internal standards, respectively. Calibration curves were constructed by linear regression analysis of the ratios of the GAA or creatine calibrator to the respective internal standard. The calibration curves (0 –500 mol/L) covered the range of GAA and creatine concentrations typically found in urine (when diluted 10-fold) or plasma (8 ). We monitored intraday (n ⫽ 5) precision of the calibration curves. The mean slope, intercept, and coefficient of linear regression for creatine were 0.039 (95% confidence interval, 0.038 – 0.040), 0.047 (0.017– 0.077) mol/L, and 0.998, respectively. The mean slope, intercept, and coefficient of linear regression for GAA were 0.037 (95% confidence interval, 0.035– 0.038), ⫺0.003 (⫺0.039 to 0.033) mol/L, and 0.998, respectively. The intraday imprecision (CV) of the global procedure (extraction and quantification) was estimated on one sample extract diluted 10-fold. The CVs were 10% for GAA and 8% for creatine in urine and 20% for GAA and 3% for creatine in plasma. The intraday CVs for the single LC/MS/MS quantification step (10 measurements of the same sample extract in the same run) were 4% for GAA and 3% for creatine in urine and 9% for GAA and 2% for creatine in plasma. Interday imprecision was assessed on the same urine and plasma samples extracted and injected on 5 days. The CVs were 10% for GAA and 9% for creatine in urine and 13% for GAA and 9% for creatine in plasma. The limit of detection (signal-to-noise ⬎3) and limit of quantification (signal-to-noise ⬎10) were measured on successive aqueous dilutions of GAA and creatine solutions. The limits of detection were 0.01 mol/L for creatine and 0.025 mol/L for GAA. The limits of quan-
tification were 0.05 mol/L (CV ⫽ 7%; n ⫽ 5) for creatine and 0.1 mol/L (CV ⫽ 6%; n ⫽ 5) for GAA. To estimate linearity, we added 25, 50, 125, 250, 500, 1250, and 2500 mol/L GAA and creatine to controls. For urine, the assay was linear for creatine between 0 and 1250 mol/L and for GAA between 0 and 2500 mol/L in a 10-fold diluted sample, corresponding to 12 500 and 25 000 mol/L, respectively, in the original sample. In plasma, the assay was linear between 0 and 1250 mol/L for creatine and between 0 and 500 mol/L for GAA. To estimate recovery, we added two concentrations of GAA and creatine (n ⫽ 5 for each concentration) to urine
and plasma. The recovery in urine of 250 and 500 mol/L GAA was 85% (CV ⫽ 14%) and 78% (CV ⫽ 14%), respectively. The recovery in urine of 250 and 500 mol/L creatine was 96% (CV ⫽ 7%) and 89% (CV ⫽ 5%), respectively. In plasma, the recovery of 20 and 40 mol/L GAA was 131% (CV ⫽ 8%) and 120% (CV ⫽ 8%), respectively, and recovery of 100 and 200 mol/L creatine was 115% (CV ⫽ 4%) and 124% (CV ⫽ 8%), respectively. We analyzed 35 urine and 21 plasma samples with the described LC/MS/MS method and a stable-isotope-dilution GC/MS method (3 ). Deming regression and Bland– Altman plots (Fig. 1) revealed excellent agreement be-
Fig. 1. Comparison of the LC/MS/MS method with a GC/MS method for urine and plasma. (A), creatine in urine (mmol/mol creatinine; n ⫽ 35); (B), creatine in plasma (mol/L; n ⫽ 21); (C), GAA in urine (mmol/mol creatinine; n ⫽ 35); (D), GAA in plasma (nmol/L; n ⫽ 21). The solid lines indicate the Deming regression; the dashed lines indicate the lines of unity. The insets show the Bland–Altman plots of the differences vs the means of paired values for the LC/MS/MS method and the GC/MS method. For creatine in urine and plasma, the regression equations were as follows (values in parentheses are the SE): urine, y ⫽ 1.11 (0.06)x ⫹ 1.58 (4.88) mmol/mol creatinine (r ⫽ 0.98); plasma, y ⫽ 1.13 (0.08)x ⫺ 1.91 (4.85) mol/L (r ⫽ 0.91). The mean differences were 67.3 mmol/mol creatinine for urine and 7.81 mol/L for plasma. For GAA in urine and plasma, the regression equations were as follows: urine, y ⫽ 1.13 (0.11)x ⫹ 0.92 (2.6) mmol/mol creatinine (r ⫽ 0.96); plasma, y ⫽ 0.87 (0.36)x ⫹ 0.79 (0.47) mol/L (r ⫽ 0.42). The mean differences were 951 mmol/mol creatinine for urine and 0.58 mol/L for plasma.
Clinical Chemistry 50, No. 8, 2004
tween the two methods for creatine and GAA in urine and creatine in plasma. For GAA in plasma, the less satisfying results are probably attributable to a lack of sensitivity of our tandem spectrometer. To assess the ability to detect CDS patients, we analyzed a urine sample from a 21-year-old patient with GAMT deficiency and compared the results with the values for controls. Reference values were estimated by use of control urines (n ⫽ 47; age range, 5–23 years of age) with nonparametric analysis (5th and 95th percentiles). As expected, urinary GAA was increased (386 mmol/mol creatinine; values for control group, 7– 88 mmol/mol creatinine), and urinary creatine was in the range of values obtained for the controls (34 mmol/mol creatinine; values for control group, 12–585 mmol/mol creatinine). In summary, we have described and validated a LC/ MS/MS method for the diagnosis of CDS-affected patients that quantifies GAA and creatine in urine and plasma by use of stable-isotope-labeled creatine and GAA as internal standards. This analytical method shows advantages of speed, specificity, linearity over large concentration ranges, and comparability with a stable-isotopedilution GC/MS method.
We thank Dr. Maria Luis Cardoso from Instituto de Genetica Medica Jacinto de Magalhaes (Porto, Portugal) for providing a urine sample from a patient with GAMT deficiency.
References 1. Schulze A. Creatine deficiency syndromes. Mol Cell Biochem 2003;244:143– 50. 2. Schulze A, Mayatepek E, Rating D, Bremer HJ. Sakaguchi reaction: a useful method for screening guanidinoacetate-methyltransferase deficiency. J Inherit Metab Dis 1996;19:706. 3. Struys EA, Jansen EEW, Ten Brink HJ, Verhoeven NM, Van der Knaap MS, Jakobs C. An accurate stable isotope dilution gas chromatographic-mass spectrometric approach to the diagnosis of guanidinoacetate methyltransferase deficiency. J Pharm Biomed Anal 1998;18:659 – 65. 4. Fingerhut R. Stable isotope dilution method for the determination of guanidinoacetic acid by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 2003;17:788 –93. 5. Carducci C, Birarelli M, Leuzzi V, Carducci C, Battini R, Cioni G, et al. Guanidinoacetate and creatine plus creatinine assessment in physiologic fluids: an effective diagnostic tool for the biochemical diagnosis of arginine: glycine amidinotransferase and guanidinoacetate methyltransferase deficiencies. Clin Chem 2002;48:1772– 8. 6. Bodamer OA, Bloesch SM, Gregg AR, Stockler-Ipsiroglu S, O’Brien WE. Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry. Clin Chim Acta 2001;308:173– 8. 7. Piraud M, Vianey-Saban C, Petritis K, Elfakir C, Steghens JP, Morla A, et al. ESI-MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode. Rapid Commun Mass Spectrom 2003;17:1297–311. 8. DeGrauw TJ, Salomons GS, Cecil KM, Chuck G, Newmeyer A, Schapiro MB, et al. Congenital creatine transporter deficiency. Neuropediatrics 2002;33: 232– 8. DOI: 10.1373/clinchem.2004.034538
Ki-67 Protein Concentrations in Urothelial Bladder Carcinomas Are Related to Ki-67-Specific RNA Concentrations in Urine, Tim B. Menke,1 Katrin Boettcher,1 Stefan Kru¨ ger,2 Ingo Kausch,3 Andreas Boehle,4 Georg Sczakiel,1 and Jens M. Warnecke1* (1 Institut fuer Molekulare Medizin, 2 Institut fuer Pathologie, and 3 Klinik und Poliklinik fuer Urologie, UK-SH, Campus Luebeck and Universitaet zu Luebeck, Luebeck, Germany; 4 HELIOS Agnes Karll Krankenhaus, Bad Schwartau, Germany; * address correspondence to this author at: Institut fuer Molekulare Medizin, Universitaet zu Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany; fax 49-451-500-2729, e-mail [email protected]
) The Ki-67 protein is a nuclear and nucleolar protein that is strictly associated with cell proliferation. Recently it has been suggested to play a role in the control of the higher order chromatin structure (1 ). Because the protein is produced only in dividing cells, the anti Ki-67 antibody MIB-1 has been widely used in histopathologic studies to estimate the growth fraction of human neoplastic tissue samples in situ. For a variety of human tumors, including bladder carcinomas, the Ki-67 labeling index has been shown to be of prognostic value for tumor recurrence and for patient survival (2– 4 ). We investigated whether Ki-67 RNA in total urine of bladder tumor patients is correlated with the Ki-67 labeling index of the corresponding tumor tissue. Spontaneously voided clean-catch urines from 68 patients were collected with informed consent, stored at 4 °C, and processed within 4 h of collection. Approval was obtained from the Ethikkommission of the University of Luebeck. Three groups of patients were included: (a) healthy donors (n ⫽ 14); (b) patients with urinary tract infection as detected by analysis of urine sediment (n ⫽ 28); and (c) patients with bladder carcinoma (n ⫽ 26). All urine samples from patients with tumors were checked for significant bacteriuria or leukocyturia (⬎10/L) as well as for the presence of erythrocytes (⬎10/L). The specific weights of all urine samples were in the range 1.010 –1.015 kg/L, showing that there were no major differences in urine concentration. After routine diagnostics were completed, 1 mL of urine was mixed 1:1 with lysis buffer [5.64 mol/L guanidinium thiocyanate, 5 g/L sarcosyl, 50 mmol/L sodium acetate (pH 6.5), 1 mmol/L ␤-mercaptoethanol], the pH was adjusted to 7 by addition of 1.5 mol/L HEPES (pH 8.0), and samples were frozen at ⫺80 °C until RNA extraction was performed. RNA was isolated by use of the RNeasy Midi reagent set (Qiagen), according to the manufacturer’s instructions except for the fact that lysis buffer (see above) was used instead of buffer RLT. We subjected 200 L of the product from the RNeasy extraction procedure to DNase I digestion, inactivated the DNase by incubation for 10 min at 65 °C in the presence of 2.5 mmol/L EDTA, precipitated the RNA in ethanol, and resuspended the pellet in 7 L of deionized water. The complete isolate was reverse-transcribed with use of random hexamer priming and superscript II reverse transcriptase (Invitrogen) according to the