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Serum carnosinase activity in plasma and serum: validation of a method and values in cardiopulmonary bypass surgery.

Serum carnosinase (EC 3.4.13.20) is synthesized in the brain, where it is secreted into cerebrospinal fluid and then into the systemic circulation. Its deficiency has been associated with neurologic deficits (1, 2), and decreased concentrations have been observed in patients with Parkinson disease or multiple sclerosis and in patients after a cerebrovascular accident (3). It has also been suggested that measurement of serum carnosinase together with neuron-specific enolase, or their ratio, may be useful in the assessment of patients with acute stroke with respect to diagnosis and prediction of clinical outcome (4).

Carnosinase exists in two isoforms (5). Tissue carnosinase is found in the liver, kidney, and spleen, and serum carnosinase is found in serum (plasma), as well as in the brain and spinal fluid (6). These enzymes result from different gene products and differ not only in their distribution but also in their enzymatic properties (7-9). Specifically, although under appropriate conditions both isoforms catalyze the hydrolysis of the dipeptide carnosine ([beta]-alanyl-L-histidine), only serum carnosinase is able to hydrolyze homocarnosine ([gamma]-aminobutyryl-L-histidine) (2, 9,10).

It is believed that carnosine and homocarnosine have the ability to protect neuronal cells against ischemic injury (11) and oxidative stress (12,13) as well as to increase their resistance toward functional exhaustion and accumulation of senile features (14).

Ischemic-reperfusion injury is a major adverse event that causes morbidity and mortality after cardiac surgery, especially when extracorporeal circuits are used. Cerebral damage has been correlated to this type of injury and leads to a decrease in neurocognitive function (15). Currently, the possible relationships between cerebral damage, neurocognitive function, carnosine and homocarnosine concentrations, and serum carnosinase activity during (open) heart surgery are not well understood.

Several methods have been described to determine carnosinase activity (3, 5, 7, 8,16), based on the reaction of o-phthalaldehyde (OPA) with histidine, which is liberated from the substrate (17,18). In this study, we used a modified version of this method to ensure that only serum carnosinase was determined and not tissue carnosinase. Samples (10 [micro]L) diluted in assay buffer (Tris-HCl, pH 7.6) were incubated for 2 min at 37[degrees]C in the presence of cobalt ions (3.0 mmol/L), after which homocarnosine (30 mmol/L) prewarmed to 37[degrees]C was added. After a 20-min incubation at 37[degrees]C, trichloroacetic acid (0.6 mol/L) was added to stop the reaction. The formed histidine was quantified by reaction with OPA to produce a fluorescent product, which was directly proportional to serum carnosinase activity. Before the histidine determination, the tubes were centrifuged at room temperature for 1 min at 13 250g. For each sample a control value was determined with the same procedure, except with the addition of trichloroacetic acid before the addition of homocarnosine to inhibit its hydrolysis. Samples (70 [micro]L) were pipetted in duplicate into the wells of a 96-well microplate and incubated for 15 min at 30[degrees]C with OPA (2 g/L) under alkaline conditions. The fluorescent adduct formed was stabilized by addition of ortho-phosphoric acid (4 mol/L). The formed complex was measured fluorometrically with excitation at 355 run and emission at 450 run. Serum carnosinase activity was expressed as the concentration of histidine formed per minute per milliliter of sample (nmol histidine x [min.sup.-1] x [mL.sup.-1]).

In preliminary tests we observed that nonhydrolyzed homocarnosine contributed to fluorescence and that calibration curves prepared in sample buffer showed slightly higher fluorescence values compared with calibration curves prepared in either heparin plasma or serum. We therefore diluted the calibrants with heparin plasma and measured the histidine fluorescence in the presence of a final concentration of 5.0 mmol/L homocarnosine. Calibrants ranged from 1 to 40 nmol of histidine, and the data were plotted on a logarithmic scale and analyzed by linear regression according to the equation: log y = a * log x + b, where y is the measured fluorescence (expressed as arbitrary units obtained with the same instrument settings throughout), and x is the amount of histidine present in nmol. Correlation coefficients were >0.99. The calibration parameters a and b were reproducible (CV <6%; n = 10), and the difference between back-calculated and nominal concentrations of the calibrators was <10%. Release of 1 nmol of histidine (the amount present in the lowest calibrant) corresponded to a serum carnosinase activity of 5 nmol histidine x [min.sup.-1] x [mL.sup.-1]. Thus the latter value was defined as the lower limit of quantification.

We investigated the influence of anticoagulants on enzymatic activity by incubating 25 [micro]L of heparin plasma with 25 [micro]L of sodium citrate to obtain final citrate concentrations ranging from 0 to 15 g/L, after which the assay procedure was carried out. Blood collected from healthy volunteers was used to prepare platelet-poor plasma and serum according to standard procedures. Heparin (1 IU/mL) was used as an anticoagulant. We observed that serum carnosinase activity was inversely related to sodium citrate concentration. At sodium citrate concentrations >5 g/L, serum carnosinase activity was approximately one-third of the initial activity. A similar reduction in activity has been reported for EDTA plasma (8). Additionally, serum carnosinase activity was studied in triplicate in serum and heparinized plasma, obtained one after the other at the same time of blood collection, from four volunteers. The results showed that serum carnosinase activities in heparin plasma and serum were similar: mean (SD) of 64.1 (5.9) nmol histidine x [min.sup.-1] x [mL.sup.-1] in plasma and 63.8 (9.1) nmol histidine x [min.sup.-1] x [mL.sup.-1] in serum. Consequently, all analyses were performed with heparin plasma in the absence of chelators or in serum. Up to 10% hemolysis had no effect on serum carnosinase activities.

We investigated assay linearity by preparing serial dilutions of heparin plasma and serum in 50 mmol/L Tris, pH 7.6, and subsequently measuring serum carnosinase activity. The slopes obtained by linear regression were close to unity (0.98 for both plasma and serum), and the y-intercepts were -0.1 nmol x [min.sub.-1] x [mL.sup.-1] for plasma and -0.8 nmol x [min.sup.-1] x [mL.sup.-1] for serum on a scale ranging up to 100 nmol x [min.sup.-1] x [mL.sup.-1] (R = 0.9997 for plasma and 0.9996 for serum). The serum carnosinase assay was linear between 8 and 100 nmol x [min.sup.-1] x [mL.sup.-1], and samples exceeding an activity of 100 nmol x [min.sup.-1] x [mL.sup.-1] could be diluted up to eightfold before analysis.

Stability was tested in duplicate in a plasma sample incubated at 37[degrees]C for up to 24 h. Up to 8 h the activity remained within 97.5% of the initial value; thereafter, the activity decreased slowly. There therefore was no instability during the time required to perform the assay. Regarding imprecision and accuracy, quality-control samples were prepared at low (3.7 nmol), medium (25.3 nmol), and high (33.4 nmol) concentrations of histidine diluted in plasma. These samples were measured in four separate batches (interbatch variability). In each batch these quality-control samples were assayed five times (intrabatch variability), and histidine was measured in duplicate. The accuracy was expressed as the difference relative to the nominal concentrations. Differences varied between -1.7% (for the highest quality-control samples) and 19.4% (for the lowest quality-control samples). The imprecision (CV) of the method was described in terms of intraassay (within run) variance and interassay (between runs) variance. Intraassay variance was <4% at all concentrations, and the interassay variances were <9%.

Clinical samples were collected at the Academic Hospital of Groningen, the Academic Hospital of Rotterdam, and the Catharina Hospital of Eindhoven after Medical Ethics Committee approval and informed consent was received from patients. Six samples were collected from 22 cardiopulmonary bypass (CPB) patients at the following time points: preoperatively (serum); during surgery at the time points pre-CPB, 30 min after start of CPB, and at the end of CPB (heparin plasma); and postoperatively at the time points 24 h and 7 days (serum). All samples were stored at -80[degrees]C until use. Before analysis, samples were thawed at room temperature, and aliquots were prepared. Clinical sample data were accepted when CV values of duplicates were <10% except for concentrations at the lower limit of quantification, where a CV of 15% was accepted.

The serum carnosinase data are summarized in Fig. 1. The results show that there was a substantial decrease in serum carnosinase activity at the onset and during CPB surgery. Just before CPB, after anesthesia, heparinization, opening of the thoracic cavity, and administration of any additional drugs, the mean serum carnosinase activity decreased by 35%. At 30 min after the start of CPB, the mean serum carnosinase activity decreased further, to 42% of the mean preoperative result. This latter decrease in activity from pre-CPB to 30 min after the start of CPB was influenced by hemodilution attributable to the connection to the heart-lung machine containing extracorporeal priming fluid. At later time points the activity showed an upward trend toward reference values.

[FIGURE 1 OMITTED]

In summary, the validated method described here is a robust approach to measuring serum carnosinase activities in serum and heparin plasma. Serum carnosinase activity in patients undergoing heart surgery showed a significant decrease. Further studies are required to investigate the implications of these results for brain damage and cognitive function during CPB. In particular, studies are needed to test the hypothesis that decreases in serum carnosinase are a functional mechanism to protect against ischemic brain damage.

DOI: 10.1373/clinchem.2003.019398

We thank Dr. I. James for valuable discussions.

References

(1.) Lenney JF, Peppers SC, Kucera CM, Sjaastad O. Homocamosinosis: lack of serum carnosinase is the defect probably responsible for elevated brain and CSF homocamosine. Clin Chim Acta 1983;132:157-65.

(2.) Willi SM, Zhang Y, Hill JB, Phelan MC, Michaelis RC, Holden KR. A deletion in the long arm of chromosome 18 in a child with serum carnosinase deficiency. Pediatr Res 1997;41:210-3.

(3.) Wassif WS, Sherwood RA, Amir A, Idowu B, Summers B, Leigh N, et al. Serum carnosinase activities in central nervous system disorders. Clin Chim Acta 1994;225:57-64.

(4.) Butterworth RJ, Wassif WS, Sherwood RA, Gerges A, Poyser KH, Garthwaite J, et al. Serum neuron-specific enolase, carnosinase, and their ratio in acute stroke. An enzymatic test for predicting outcome? Stroke 1996;27:2064-8.

(5.) Murphey WH, Patchen L, Lindmark DG. Camosinase: a fluorometric assay and demonstration of two electrophoretic forms in human tissue extracts. Clin Chim Acta 1972;42:309-14.

(6.) Jackson MC, Kucera CM, Lenney JF. Purification and properties of human serum carnosinase. Clin Chim Acta 1991;196:193-205.

(7.) Lenney JF, George RP, Weiss AM, Kucera CM, Chan PWH, Rinzler GS. Human serum carnosinase: characterization, distinction from cellular carnosinase, and activation by cadmium. Clin Chim Acta 1982;123:221-31.

(8.) Lenney JF, Peppers SC, Kucera-Orallo CM, George RP. Characterization of human tissue carnosinase. Biochem J 1985;228:653-60.

(9.) Teufel M, Saudek V, Ledig J, Bernhardt A, Boularand S, Carreau A, et al. Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase. J Biol Chem 2003;278:6521-31.

(10.) Pegova A, Abe H, Boldyrev A. Hydrolysis of carnosine and related compounds by mammalian camosinases. Comp Biochem Physiol B Biochem Mol Biol 2000;127:443-6.

(11.) Tabakman R, Lazarovici P, Kohen R. Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia. J Neurosci Res 2002;68:463-9.

(12.) Kang JH, Kim KS, Choi SY, Kwon HY, Won MH, Kang TC. Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu, Zn-superoxide dismutase modification. Biochim Biophys Acta 2002; 1570:89-96.

(13.) Boldyrev A, Song R, Lawrence D, Carpenter DO. Camosine protects against excitotoxic cell death independently of effects on reactive oxygen species. Neuroscience 1999;94:571-7.

(14.) Boldyrev AA. Problems and perspectives in studying the biological role of carnosine. Biochemistry (Moscow) 2000;65:751-6.

(15.) Newman MF, Kirchner JL, Phillips-Bute B, Gaver V, Grocott H, Jones RH, et al. Longitudinal assessment of neurocognitive function after coronary-artery bypass surgery. N Engl J Med 2001;344:395-402.

(16.) Orfanos AP, Guthrie R, Jinks DC. A fluorometric micromethod for estimation of carnosinase in dried blood samples. Clin Chim Acta 1987;166:219-25.

(17.) Chen RF, Scott C, Trepman E. Fluorescence properties of o-phthaldialdehyde derivatives of amino acids. Biochim Biophys Acta 1979;576:440-5.

(18.) Yoshimura T, Kamataki T, Miura T. Difference between histidine and histamine in the mechanistic pathway of the fluorescence reaction with ortho-phthalaldehyde. Anal Biochem 1990;188:132-5.

Pieter Schoen, * Hilco Everts, Theo de Boer, and Wim van Oeveren (HaemoProbe BV, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands; * author for correspondence: fax 31-50-3176790, e-mail pieterschoen@ haemoprobe.com)
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Title Annotation:Technical Briefs
Author:Schoen, Pieter; Everts, Hilco; de Boer, Theo; van Oeveren, Wim
Publication:Clinical Chemistry
Date:Nov 1, 2003
Words:2152
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