Application of at-line two-dimensional liquid chromatography–mass spectrometry for identification of small hydrophilic angiotensin I-inhibiting peptides in milk hydrolysates

A two-dimensional chromatographic method with mass spectrometric detection has been developed for identification of small, hydrophilic angiotensin I-inhibiting peptides in enzymatically hydrolysed milk proteins. The method involves the further separation of the poorly retained hydrophilic fraction from a standard C18 reversed-phase column on a hydrophilic interaction liquid chromatography (HILIC) column. The latter column is specifically designed for the separation of hydrophilic compounds. Narrow fractions collected from the HILIC column were analysed for their angiotensin I-converting enzyme (ACE) inhibiting potential in an at-line assay. Fractions showing significant inhibition of ACE were analysed by LC–MS for structure elucidation. With this method the main peptides responsible for ACE-inhibition in the hydrophilic part of a milk hydrolysate could be determined. The ACE-inhibiting peptides RP, AP, VK, EK, and EW explained more than 85% of ACE-inhibition by the hydrophilic fraction.
Introduction
Hypertension is one of the major health problems in the western world. A variety of food ingredients that help to reduce blood pressure have been described. Particularly attractive are hydrolysed proteins from fermented milk containing specific peptides that can inhibit the angiotensin I-converting enzyme (ACE), an enzyme involved in blood-pressure regulation [1–3]. For the development of optimized anti-hypertensive food products, it is crucial to know which specific peptide(s) in the complex protein hydrolysate is (are) responsible for the ACE-inhibition.
Identification of active ingredients in complex mixtures is generally done using assay-guided repeated fractionation. For the specific case of ACE-inhibiting peptides in milk-protein hydrolysates often only part of the total activity of the product can be assigned to specific peptides [4]. A significant part of the remaining activity is present in fractions that are poorly retained by the standard reversed-phase columns used in the fractionation procedure. Nakamura et al. [1] for example could only explain part of the activity of a casein hydrolysate by the tri-peptides VPP and IPP eluting in one of the later fractions. The remaining activity was present in the unretained fraction. In our laboratory similar samples were seen to have over 30% of the total activity in the unretained fraction. Identification of the oligopeptides in this fraction was not further pursued but would have been extremely difficult because the fraction contains far too many compounds for unambiguous activity assignment. Moreover, there are also too many interfering compounds present in the first-eluting fractions for reliable MS identification. Similar situations are also described by other authors [5–7]. In a previous investigation we presented a new at-line method for the identification of ACE-inhibiting (oligo)peptides in hydrolysed caseinate [4]. Approximately 70% of the activity of these samples was caused by peptides nicely retained by the two reversed-phase columns used in the fractionation. Twenty percent of the activity was present in the hydrophilic fraction. The remaining 10% was caused by a large number of peptides with low ACE-inhibiting activity. Identification of the peptides in the hydrophilic fraction was impossible due to the large number of peptides present and the presence of co-eluting compounds, such as salts and sugars, that resulted in severe suppression of ionisation. Two reversed-phase columns were used in an at-line 2D set-up to provide improved resolution of the very complex mixture. Unfortunately, the highly hydrophilic peptides escaped identification since they were unretained on both columns. To identify the peptides responsible for the ACE inhibition of the unretained hydrophilic fraction improved chromatographic retention and separation is required. Hydrophilic interaction chromatography (HILIC) could be an attractive way of achieving this.
The use of HILIC for separation of hydrophilic substances such as proteins, peptides, and nucleic acids was first described by Alpert in 1990 [8]. Since then HILIC has been used for the separation of many different substances, for example denaturants [9], polar oligomers [10], and pharmaceutical ingredients [11]. Many publications can be found describing the increased retention of hydrophilic peptides on HILIC columns in comparison with reversed-phase columns. A detailed review is published by Yoshida [12]. Literature describing the application of reversed-phase separation in combination with HILIC chromatography is scarce. A notable exception is the compositional analysis of an enzymatic protein hydrolysate obtained from acid-deaminated wheat gluten by Schlichtherie-Cerny [13]. The hydrolysate was first fractionated by GPC. The fraction corresponding to a molecular weight range of 100–750Da was then further fractionated by RPLC. Finally, the peptides in the unretained peak from the RPLC column were separated and characterised by HILIC–API-MS. Using this method ten amino acids, eight di-peptides and one tri-peptide, were identified in this fraction.
In this paper we describe the development of a method for the identification of ACE inhibiting peptides in the poorly retained fractions of a reversed-phase fractionation of an enzymatically hydrolysed milk protein. The possibility of improving chromatographic resolution by using a HILIC separation as a second-dimension separation after the first reversed-phase isolation is studied. The aim of the new method is to extend the applicability of a previously developed system for the identification of ACE-inhibiting peptides in complex peptide mixtures such as protein hydrolysates [4]. The applicability of the method is demonstrated by the identification of hydrophilic ACE-inhibiting peptides in an enzymatically hydrolysed milk protein.
Experimental
Chemicals
The model peptides VPP, IPP, and LPP (purity >98%) were purchased from Bachum (Dübendorf, Switzerland). All other peptides were synthesised by JPT Peptide Technologies (Berlin, Germany). For HPLC analysis, acetonitrile, formic acid, and trifluoroacetic acid were purchased from Merck (Amsterdam, The Netherlands). Aqueous ammonia (Merck) was used for neutralization of the HPLC fractions. For instrument tuning PEG 300, PEG 600, and PEG 1000 were purchased from Sigma Chemicals (St Louis, MO, USA). Ammonium acetate was obtained from Merck. For the at-line assay angiotensin I-converting-enzyme (ACE) and hippuryl–histidyl–leucine (HHL) were purchased from Sigma–Aldrich Chemie (Zwijndrecht, The Netherlands). PBS buffer was purchased from Gibco (Paisley, UK). The hydrolysed milk powder was an enzymatically hydrolysed milk protein obtained from Calpis (Tokyo, Japan).
Instrumentation
All peptide separations and identifications were performed with a Waters Alliance 2795 HT HPLC coupled to a Micromass QTOF-Ultima hybrid time-of-flight mass spectrometer equipped with a lock spray option for accurate mass determination (Waters, Almere, The Netherlands). Fractions were collected in polypropylene 300-μL 96-well plates (Nunc, Roskilde, Denmark) using a Mark IV fraction collector also from Waters. Solvent evaporation was performed using an Ultravap 96-well evaporation device (Porvair, Shepperton, UK). Quantification of HHL and HL in the at-line assay was performed on the QTOF instrument described above.
Two-dimensional analytical separation
The first-dimension reversed-phase separation was performed on two serially connected 150 × 2.1mm Inertsil 5 ODS3 columns, particle size 5μm, operating in the gradient mode (Varian, Middelburg, The Netherlands). The guard column was a 20 × 3.9mm SymmetryShield RP8 column packed with 5-μm particles (Waters, Etten-Leur, The Netherlands). Mobile phase A consisted of 0.1% TFA in Milli-Q water. Mobile phase B consisted of 0.1% TFA in acetonitrile. The initial eluent composition was 100% A. After a 5min hold a linear gradient was started to 5% B in 15min, followed by a linear gradient to 70% B in 75min. Finally, mobile phase composition was programmed to 99% B in 1min and kept at 99% B for another 4min. At the end of the gradient the eluent composition was reduced to 100% A in 6min and the system was allowed to re-equilibrate for 14min. The total run time was 120min. The eluent flow rate was 0.2mL min−1 and the column temperature was 60°C. In-house experiments revealed that at this temperature isomeric peptides such as IPP and LPP are separated. UV detection was performed at 215nm and 254nm. Fractions of 200μL were collected in 96-well plates. After neutralization and solvent evaporation the residues were reconstituted in 50μL of an 80:20 mixture of solvents A and B for the second-dimension HILIC analysis.
The second dimension HILIC analysis was performed on a 150 × 2.1mm HILIC Atlantis column with a particle size of 3μm (Waters, Etten-Leur, The Netherlands). Here solvent A consisted of 0.1% formic acid in acetonitrile and solvent B of 10mmol L−1 aqueous ammonium acetate solution+0.1% formic acid in Milli-Q water. The separation was performed at 40°C, the maximum temperature specified by the manufacturer, and a flow rate of 0.2mL min−1. The initial eluent composition was 95% A. A linear gradient was used to 70% B in 30min, followed by a linear gradient to 90% B in 5min where it was kept for another 5min. At the end of the run the eluent was reduced to 95% C in 2min. The eluent flow was then increased to 0.3mL min−1 in 1min. This flow rate was maintained for another 7min. Finally the flow was reduced to 0.2mL min−1 in 1min. and the system was allowed to equilibrate for 3min. The total run time was 54min. 45μL of each fraction collected from the ODS3 column was injected. At the outlet of the second dimension HILIC column fractions of 200μL were collected in 96-well plates. The fractions were neutralized, evaporated, and transferred to the activity assay (next section).
At-line Matsui assay
ACE inhibition by the collected fractions was measured in a 96-well plate assay according to the at-line method described in an earlier publication [1]. The method is based on the conversion of hippuryl–histidyl–leucine (HHL) into hippurate (H) and histidyl–leucine (HL) by the angiotensin I-converting enzyme (ACE). Briefly, after evaporation of the eluent the fractions were reconstituted in 50μL PBS buffer and vortex mixed for 1min. This solution (40μL) was used for the at-line assay. Percentage inhibition was calculated from the peak areas of HHL and H at 280nm. For the active fractions the remaining 10μL was used for identification of the peptides.
Structural identification of ACEI peptides
Peptide identification was performed on the QTOF MS instrument using HPLC–MS and MS–MS in the accurate mass mode. Fractions collected after the second dimension that showed activity were separated on the HILIC column now coupled to the MS using the gradient described above. The source and desolvation temperatures were 100°C and 300°C, respectively. The cone and desolvation gas flows were 100L h−1 and 800L h−1, respectively. The capillary voltage was 4kV and the cone voltage 35V. The collision energy was 10eV in full-scan mode and 25eV in MS–MS mode. The collision gas was argon and the analyser pressure was 4 × 10−5 mbar. In the lock spray probe a mixture of PEG 300, PEG 600, and PEG 1000 dissolved in 10mmol L−1 ammonium acetate in methanol–water 1:1 (v/v) at concentrations of 0.01, 0.02, and 0.04μg mL−1, respectively, was used. The reference flow was 5μL min−1. Spectra were recorded at a resolution of 10,000. Masslynx software (Waters) was used for data acquisition and evaluation.
Results and discussion
In a reversed-phase fractionation of a protein hydrolysate a significant percentage of the activity can be present in the unretained hydrophilic fraction. To be able to calculate the contribution of this fraction of our milk hydrolysate to the total activity, an ACE-inhibition profile of the entire product was produced. To this end 20μL of a 20mg mL−1 solution of the milk hydrolysate in solvent A was injected on to the 300mm ODS3 column. Fractions of 200μL each were collected from 0 to 65min, with an interval time of 1min, in a 96 well plate. The ACE inhibition of each fraction was determined. Figure 1a shows the MS TIC of the fermented milk and Fig. 1b shows the activity profile of the same analysis. From the data in Fig. 1b it was calculated that the hydrophilic compounds in fractions 6 to 15 represent approximately 25% of the total ACE inhibition. MS–MS showed that the high activity in fractions 27 to 31 resulted from the known ACE inhibiting peptides IPP and LPP. The activity in fractions 21 to 23 resulted from the peptide VPP. These peptides have been identified to be the main contributors to the ACE-inhibition of a Lactobacillus helveticus-fermented milk [2] and represent 40% of the total ACE inhibition. The remaining activity between fractions 16 and 40 is most likely caused by a mixture of ACE-inhibiting peptides of different chain lengths. This is still under investigation. In the region from fraction 6 to fraction 15 a large number of peptides co-elute and the spectra of the fractions are highly complex. Figure 2, for example, shows the mass spectrum of fraction 7 collected from the first dimension ODS 3 columns and the ion traces of three of the ACE-inhibiting peptides discussed later in this manuscript. At least 16 ions can be observed with intensities above 10% of the base peak, all representing different peptides. In order to be able to assign the measured activity to one or more of these peptides a further separation in an additional chromatographic dimension is required. To achieve this, fractions collected from the first-dimension ODS column were injected on the second dimension HILIC column. Again 200-μL fractions were collected in 96-well plates. After neutralization and evaporation the residues were reconstituted in 50μL Milli-Q water. Of this, 40μL was used for ACE inhibition testing, the remaining 10μL for MS identification. As an example of the results of these experiments, Fig. 3 shows the TIC chromatogram obtained from full-scanning analysis of fraction 7 of the ODS3 columns, now separated on the second dimension HILIC column. The TIC chromatogram clearly shows that the composition of this fraction is indeed very complex. To identify the peptide(s) responsible for the ACE-inhibition, the fractions collected in the 96-well plate were subjected to the ACE-inhibition assay. Figure 4 displays the activity distribution of ODS fraction 7. The data in Fig. 4 are corrected for the background recorded using a blank gradient, i.e. a gradient without injection. Blank analysis of the HILIC column showed slightly negative values compared with a reference of pure PBS. The reason for these negative responses is still unknown. The results in Fig. 4 clearly show the strong activity in HILIC fractions 17 to 19 and somewhat lower activity in fractions 12 to 16 from the HILIC column. LC–MS separations were performed on these fractions and the MS data were searched for peptides in the two active regions. In Fig. 5 the mass spectrum of fraction 18 is given. In the spectrum three abundant ions can be observed. A library search and MS–MS measurements revealed that m/z 272.1721 represents the molecular ion of the di-peptide RP (−0.7ppm), while m/z 253.1190 represents the molecular ion of HP (−2.1ppm) and m/z 235.1305 the loss of water from m/z 253.1190. Identification of the peptides in the remaining fractions of the HILIC column showed that fraction 7 of the first-dimension ODS3 column consisted of three free amino acids, 19 di-peptides and 2 tri-peptides while six compounds remained unidentified. The positively identified amino acids were E, Q, and K and the di-peptides were QD, KY, ER, RE, KP, HP, RP, AP, VK, EK, EW, and PH. The di-peptides ET, TP, TQ, PQ, KV, KE, and HK and the tri-peptides APK and VRG were tentatively identified. Structure confirmation was based on elution time, measured exact mass (error <5ppm) and MS–MS fragmentation pattern compared with those of the model compounds. Using this method the remaining hydrophilic fractions 5 to 15 collected from the ODS3 column were also analysed in the 2D mode. In Fig. 6 a three-dimensional plot of the activity distribution over the fractions of the two columns is given. In total, five amino acids, 35 di-peptides, 13 tri-peptides, one penta-peptide and 18 not yet identified compounds were found. The identities of all amino acids and the sequence of 27 di-peptides were again confirmed by use of model compounds. Table 1 lists the sequences of the identified peptides together with their reported IC50 values and ACE inhibition data at 20μmol L−1 established in house (Foltz et al. manuscript in preparation). For peptides for which no model compounds were available, identification was based solely on the fragmentation pattern in MS–MS.
Fig. 1LC separation of a 20 mg mL−1 solution of the milk hydrolysate powder on the ODS-3 reversed-phase column. a MS-TIC chromatogram. b Activity profile. Error bars indicate the standard deviation (n = 3). Fraction volume 200 μLFig. 2Mass spectrum of fraction 7 collected from the first-dimension ODS3 columnFig. 3MS-TIC of fraction 7 from the ODS3 column analysed on the HILIC column. The inset shows the selected ion traces of three of the most active peptidesFig. 4ACEI profile of fraction 7 from the ODS3 column analysed on the HILIC columnFig. 5Mass spectrum of fraction 18 collected from the HILIC columnFig. 6Three-dimensional display of the ACEI distribution of the fractions collected from the ODS3 column and the HILIC columnTable 1Peptides identified in fractions 6 to 15 of the ODS3 column, analysed on the HILIC columnelution (min) timepeptide sequenceIC50 value (μM) literatureLiterature referenceInhibition at 20 μM (%)peak area (counts)elution time (min)peptide sequencevalue (μM) literatureLiterature referenceInhibition 20 μM (%)peak area (counts)11.64Fn.r.  8316.34FP1215.713n.m.63111.90Yn.r.  227416.41HSMn.r.  65612.02EIn.r. 38.2222116.43EPFn.r.  18612.36IEn.r. 3.7459116.43DKIn.r.  18712.40YP72013n.m.5816.52KY1.63,7.8,1316,13,17n.m.1312.59EVn.r. 25.462216.66VYP28813 12512.59VEn.r. 41.02217.00EWn.r. 65.1157812.59IP1291317.710017.22VK12.91357.216712.84VP5751345.980117.23ERn.r. n.m.23613.21En.r. n.m.28817.30SGYn.r.  62413.26TEn.r. 26.034417.35MEn.r. 24.64213.44LQn.r. 11.274817.55Kn.r. n.m.49713.74KP16,22,3013,1533.714417.61EKn.r. 41.833713.81VNEn.r.  137817.84REn.r. n.m.16614.08LNn.r. 14.727117.86KEn.r. 13.48314.10Qn.r. n.m.51117.89KV33 n.m.23514.10QDn.r. n.m.79118.04VVRn.r.  11414.28TDVENn.r.  201918.08HPn.r. 11.1142714.50PTn.r. 3.1100118.18KVPn.r.  4514.56NVPn.r.  274418.24VPQn.r.  50514.61YQn.r. n.m.3618.63RP21,91,1821336.4134014.64SPPn.r.  33518.97PHn.r. 0.030614.72TQn.r. 0.017519.03QPn.r. n.m.40214.86AHn.r. 7.310119.11KP16,22,3013,1533.614414.91PP2284.7180.0175919.22APKn.r. n.m.26615.01AP29,26913,1011.481120.18VRGn.r. n.m.31315.18PQn.r. 0.0104120.47HKn.r. 0.0398The sequences of the bold printed peptides were confirmed by MS–MS analyses of reference peptides, n.r., not reported in the literature; n.m., not measured
To verify whether the identified peptides indeed fully explain the measured activity of the hydrophilic fraction the concentration of all peptides should be determined. Next a mixture should be prepared which is then analysed using the two-dimensional method described in this article. The ACE-inhibition profile of this mixture should be identical with that of the sample. Such an experiment will hardly be feasible because it requires the availability of all the components. To estimate of the completeness of identification, the contribution of each peptide was estimated from the measured peak area and the IC50 value obtained from literature or from unpublished in-house data. To estimate the contribution of an individual peptide the following relationship was used: CA ≈ Ap/IC50p. In this equation CA represents the contribution of the peptide to the measured ACE-inhibition, Ap the peak area, and IC50p the IC50 value of the peptide. Clearly this proportionality equation is only approximate as it assumes that the MS response for all peptides is identical. When both the peak area and the IC50 value are taken into consideration it can be concluded that the di-peptides AP, RP, VK, and EK are the main peptides responsible for the measured ACE-inhibition of the hydrophilic fractions 6 to 10 collected from the ODS3 column (Fig. 7). In Fig. 8 the activities of all peptides in each fraction are summarized and the profile is compared with that of the measured profile. The two profiles are similar. The differences between the graphs can be explained from the fact that no correction was applied for differences in MS responses of the various peptides.
Fig. 7Distribution of the ACE-inhibiting activity, calculated from Table 1, in hydrophilic fractions 5 to 10 collected from the ODS3 columnFig. 8Calculated (bottom) and measured (top) ACE inhibition profiles
The remaining fractions 11 to 15 of the ODS3 column were analysed in the same way as fractions 6 to 10. These analyses showed that the peptide EW is responsible for the major part of the ACE-inhibition measured in these fractions. In total, the five peptides identified in the fractions 6 to 15 are responsible for approximately 85% of the activity measured in the hydrophilic fractions. The most important contribution is that of the di-peptide RP, which is responsible for 34%. Together with the peptides VPP, IPP, and LPP now approximately 65% of the total activity of the product has been explained. The remaining 35% is distributed over a large number of peptides with relatively low overall activities.
When compiling the list of published IC50 values for the peptides identified in this study in some cases large differences were seen for the IC50 values of proline-containing peptides in different publications. The reported IC50 values for AP, for example, vary between 29 and 269 μmol L−1 [14]. This large difference might be caused by variations in the cis-to-trans ratio of the proline present in synthetic model compounds as a result of differences in the synthetic routes (Fmoc or Boc), as was shown for the peptide DKIHP by Gómez-Ruiz [15]. In our calculations the trans-Pro value of 29 μmol L−1 for AP was used, because trans-Pro is known to be dominant in natural products [15]. Many of the identified di-peptides were found in milk hydrolysates for the first time. For most of the newly identified ACE active peptides it is actually the first time they are reported at all. The long list of newly identified peptides found here clearly demonstrates the potential of the two dimensional separation approach of HILIC and reversed-phase HPLC described here.
Conclusions
Two-dimensional liquid chromatography in combination with mass spectrometry was successfully used for identification of poorly retained peptides present in enzymatically hydrolysed milk protein. A standard C18 reversed-phase column was used for the first separation followed by a second dimensional separation on a HILIC column. This two-dimensional procedure significantly improves the separation of hydrophilic peptides that elute almost unretained on a reversed-phase column and co-elute with numerous other compounds such as carbohydrates and salts. These compounds suppress the MS ionization and complicate identification. The method enabled the identification of hydrophilic peptides in complex mixtures. In the hydrophilic fraction of the milk hydrolysate investigated 71 compounds were found, including five free amino acids, 35 di-peptides, 12 tri-peptides, one penta-peptide, and 18 not yet identified compounds. Five peptides, RP, AP, VK, EK, and EW were responsible for approximately 85% of the measured activity of the hydrophilic fraction; of these RP made the highest contribution of 34%. None of the five peptides has, to our knowledge, been reported earlier in milk hydrolysates or related products such as yogurt or cheese. Most of the peptides involved remained undetected in single-dimensional chromatography.

reversed-phase
hilic
ms
hplc
angiotensin-converting enzyme
blood pressure-lowering peptides


Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation

Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly–Arg–Gly–Asp–Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser–Asp–Gly–Arg–Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 ± 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 ± 0.8%, p < 0.001) and control (3.0 ± 3.4%, p < 0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 ± 3.8%) equalled that from controls (15.2 ± 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p < 0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.
Introduction
Troponins are myofibrillar proteins involved in the regulation of actin–myosin interaction, thereby controlling contraction and relaxation. Cardiac troponins (cTnT, cTnT, and cTnC) are predominantly bound, via tropomyosin, to actin filaments of sarcomeres, with only a small proportion of cTnT (6–8%) and cTnI (3–8%) found in the soluble cytoplasmic pool [1]. Due to high cardiac specificity, serum concentrations of cTnI and cTnT are well-established diagnostic and prognostic markers of irreversible myocardial damage in acute coronary syndromes [9]. However, elevated serum levels of cardiac troponins have also been observed in patients without acute coronary syndromes in whom irreversible myocardial cell injury was not a prominent aspect [8, 11, 20]. Several studies reported elevated serum levels of troponins in patients with cardiomyopathy [29], heart failure [18], unstable angina pectoris [14], pulmonary embolism [17], renal insufficiency [5], and in ultra-endurance athletes [19]. The exact mechanism underlying the release of troponins in the absence of necrosis, without lethal disruption of the sarcolemma, remains to be elucidated and most likely differs from the release of troponins due to necrotic cell death.
A potential explanation for the cTnI release without lethal sarcolemmal disruption is the cellular release of proteolytic cTnI degradation products. Indeed, in isolated rat hearts, Feng et al. [6] showed degradation of cTnI upon increasing preload, which was independent of ischemia. They proposed that the cTnI degradation might be caused by increased myocardial stretch per se.
Mechanical stretch of cardiomyocytes, as occurs during pressure or volume overload, initiates a cascade of intracellular signals, including increased intracellular calcium concentration, increased intracellular NO formation, and the activation of intracellular proteases such as MMP2 and MMP14 [28, 36]. MMP2 is able to degrade cTnI intracellularly [39] and may be involved in the stretch-induced release of cTnI and its degradation products. In contrast, other studies suggest that cTnI leaks from reversibly damaged cardiomyocytes as an intact non-degraded molecule [8, 20].
Mechanical stretch of cardiomyocytes is sensed by stimulation of integrins, which are transmembrane glycoprotein receptors that link the extracellular matrix (ECM) to the intracellular cytoskeleton [26]. Integrins act as mechanotransducers and bidirectional signaling molecules and participate in overload-induced hypertrophic and post-infarct remodeling [26]. The Arg–Gly–Asp (RGD) sequence that is present in fibronectin and other ECM proteins is an agonist for integrins [27], which can be used to simulate myocardial mechanical stretch in vitro.
We hypothesize that the stretch-related process through which cTnI can be released from viable cardiomyocytes is mediated by integrin stimulation. The purpose of the present study is therefore to investigate whether (1) RGD-induced integrin stimulation causes release of intact and/or fragmented cTnI from cardiomyocytes in the absence of necrotic cell death, (2) integrin stimulation is associated with MMP2 activation and MMP2-related cTnI degradation, and (3) cTnI release in the absence of necrosis differs from the release of cTnI in the presence of necrosis.
Materials and methods
Primary cultures of neonatal cardiomyocytes
Primary cultures of cardiomyocytes were prepared from the ventricles of 2-day-old Wistar rats as described previously [24]. Briefly, ventricles were minced, and cells were isolated enzymatically using collagenase type I (CLS, Lakewood, NJ, USA) at 37°C. The cell suspension was centrifuged, and the cell pellet was resuspended in growth medium containing Ham’s F10 (Flow Laboratories, Irvine, UK), 10% fetal bovine serum, 10% horse serum (HS), 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen, Breda, The Netherlands). The cells were seeded in 6-cm diameter Primaria®-coated plastic culture dishes (Falcon, Becton Dickinson, Etten-Leur, The Netherlands), and after 45 min, non-adherent cells representing the cardiomyocytes were collected and plated in six-wells plates (∅ 35 mm per well, Costar, Corning, NY, USA) at a density of ≈1.5 × 106 cells per well. Cardiomyocytes were incubated in a humidified incubator at 37°C and 5% CO2 and culture medium [1:1 (v/v) mixture of DMEM (Invitrogen) and Ham’s F10 supplemented with 5% HS, penicillin (100 U/ml) and streptomycin (100 μg/ml)] was refreshed after 20 h and 48 h. After 3 days of culturing, a monolayer of spontaneously beating cardiomyocytes had formed. The investigations had the approval of the Animal Experiments Committee of the LUMC according to Dutch law.
Experimental protocol for integrin stimulation
Three days after cell isolation, cardiomyocyte cultures were washed twice in HEPES-buffered salt solution (HBSS), containing (in millimole per liter) NaCl (125), KCl (5), MgSO4 (1), KH2PO4 (1), CaCl2 (2.5), NaHCO3 (10), d-glucose (5), HEPES (20), pH 7.4 at 37°C. Cardiomyocytes were incubated for 24 h in 1 ml of HBSS supplemented with (1) the pentapeptide Gly–Arg–Gly–Asp–Ser containing the GRGDS sequence (Sigma, St. Louis, MO, USA) at various concentrations (100, 200, or 300 μg/ml) to stimulate integrins, with (2) Ser–Asp–Gly–Arg–Gly (SDGRG, the reverse sequence of GRGDS, Sigma), which does not affect integrin function, at a concentration of 300 μg/ml, or with (3) phosphate-buffered saline (PBS) as a control. After 24 h, medium was separated from the cells, cells were taken up in 1 ml of ice-cold lysis buffer (100 mmol/l Tris–HCl, 0.1% Tween, pH 7.5), both cell and medium samples were kept on ice awaiting assay of lactate dehydrogenase (LDH) activity on the same day, and subsequently stored at −20°C for later cTnI and MMP2 assays.
Troponin-I ELISA
Purified human heart cTnI (Calbiochem, LaJolla, CA, USA) was dissolved in urea/Tris buffer according to the manufacturer’s instructions. Monoclonal mouse anti-cTnI clone 19C7 (directed against amino acid sequence 40–50) and monoclonal mouse anti-cTnI clone 6F9 (directed against amino acid sequence 189-195) were purchased from HyTest (Turku, Finland). The enzyme-linked immunosorbent assay (ELISA) was based on the sandwich principle as described previously [13]. The percentage cTnI released from each culture equalled . Detection limit of the assay was 6 ng/ml. Inter-assay variability was 6 and 10% at a TnI concentration of 200 and 25 ng/ml, respectively. The intra-assay variability was 6% at TnI concentrations of 100 ng/ml.
Western blot analysis
Protein extracts were size-fractionated on NuPage Novex 12% Bis–Tris gels (Invitrogen) and transferred to Hybond PVDF membranes (Amersham Biosciences, Roosendaal, The Netherlands). Non-specific binding sites were blocked using blocking solution consisting of 20 g/l ECL advance blocking agent (Amersham Biosciences) in TBS-Tween (10 mmol/l Tris–HCl, 150 mmol/l NaCl, 0.05% Tween pH 8.0). Membranes were incubated for 1 h with the anti-TnI antibody (K83085G, Biodesign, Saco, ME, USA), which detects various cTnI degradation products. After four washes in TBS-Tween, membranes were incubated with horseradish peroxidase-labeled secondary antibody (rabbit anti-goat IgG, Santa Cruz Biotechnology, Heidelberg, Germany). Chemiluminescence was induced by ECL advance detection reagent (Amersham Biosciences) and detected by exposure to Hyperfilm ECL (Amersham Biosciences).
LDH activity assay
Necrotic cell death was quantified by assay of LDH activity released from cells into medium according to Wroblewski and Ladue [41]. LDH activities of medium and cell samples were determined using a spectrophotometer (Ultrospec 3000, Pharmacia Biotech, Roosendaal, The Netherlands) at 25°C. The total LDH activity in each culture, i.e., cell+medium, was determined, and the LDH activity in cell and medium samples was expressed as a percentage of total LDH activity. The detection limit of the LDH assay was 5 U/l, the intra-assay variability was 3% (110 U/l) and 6% (48 U/l), and inter-assay variability was 12% (110 U/l).
Nuclear staining with propidium iodide
An additional assessment of cell death was performed by quantifying the nuclei of necrotic cells using propidium iodide and fluorescence microscopy. For this purpose, cardiomyocyte cultures were treated with PBS, GRGDS, and SDGRG for 24 h, then incubated with propidium iodide (5 mg/l, Molecular Probes, Eugene, OR, USA) for 15 min in the dark, washed in PBS, and viewed under a fluorescence microscope (Zeiss, Hamburg, Germany) at 535/617-nm excitation/emission. Subsequently, cultures were incubated with digitonine (10 μmol/l) for 5 min to induce necrotic cell death in all cardiomyocytes. The extent of necrosis equalled the number of necrotic nuclei before digitonine incubation as a fraction of the number of necrotic nuclei after digitonine incubation (100%).
In vitro degradation of purified cTnI by active MMP2
To study whether active MMP2 is able to degrade cTnI in vitro, purified human cardiac TnI (500 pg/μl, Calbiochem) was incubated with human recombinant active MMP2 (1.5 ng/μl, Oncogene, San Diego, CA, USA) in enzyme buffer at 37°C for 0, 60, and 120 min. In a separate series of experiments, MMP2 was preincubated with an inhibitor of MMP2, o-phenanthroline (100 μmol/l) for 15 min. As a control, cTnI was incubated without active MMP2 at 37°C. Intact cTnI and cTnI degradation products were visualized by Western blotting.
Zymography
Gelatinolytic activities of MMP2 in cell and medium samples of control, GRGDS- and SDGRG-treated cardiomyocytes were analyzed by zymography as described previously [12, 33]. Briefly, cell samples (tenfold concentrated by freeze-drying for 18 h) and medium samples (unconcentrated) were prepared in sample buffer and were loaded on gels. After electrophoresis, the gels were washed twice in 2.5% Triton X-100, were washed in enzyme buffer, and were incubated in enzyme buffer at 37°C overnight. Gels were stained with amino black and subsequently destained. Gelatinase activity was quantified by measuring the extent of gelatin digestion using a scanning densitometer (2202 Ultrascan, LKB, Paramus, NJ, USA).
Experimental protocol for necrotic cell death
Three days after cell isolation, cardiomyocyte cultures were washed twice in HBSS, pH 7.4, at 37°C, and subsequently incubated in 1 ml of HBSS, supplemented with sodium azide (1 mmol/l, n = 7) at 37°C. After 24 h of incubation, medium was separated from the cells, cells were taken up in 1 ml of ice-cold lysis buffer, both cell and medium samples were kept on ice awaiting assay of lactate dehydrogenase (LDH) activity on the same day, and subsequently stored at −20°C for later cTnI assay.
Statistics
Results are expressed as mean ± SD. Statistical analysis was performed by Student’s t test and by one-way analysis of variance followed by Bonferroni’s post hoc test. Differences were regarded as statistically significant if p < 0.05. SPSS14 for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis.
Results
Effect of integrin stimulation on release of cTnI
Cell and medium samples were assayed for intact cTnI by ELISA (Fig. 1a) and for cTnI degradation products using Western blotting (Fig. 2a–d). Cellular cTnI content in ten cultures was 0.27 ± 0.10 μg per culture (100%). Control cardiomyocytes had released ≈3% of total cTnI, probably as a result of cell death of a few cardiomyocytes during 24 h of incubation. GRGDS treatment at a concentration of 100 or 200 μg/ml did not show considerable release of cTnI (≈2%). However, a concentration of 300 μg/ml GRGDS resulted in a significant extra release of intact cTnI (to ≈10%), compared with control cardiomyocytes (p < 0.001) and cardiomyocytes treated with 100 or 200 μg/ml GRGDS (both p < 0.001; Fig. 1a). Incubation of cardiomyocytes with 300 μg/ml SDGRG, the reverse sequence of GRGDS that has no effect on integrin function, did not induce a significant extra release of intact cTnI (Fig. 1a). In addition, Fig. 2b and d shows that cTnI was released as an intact protein of 29 kDa and that integrin stimulation is not associated with the release of cTnI degradation products.
Fig. 1a Release of cardiac troponin-I (cTnI) from cardiomyocytes incubated with PBS (control, n = 38), GRGDS (100 μg/ml, n = 5; 200 μg/ml, n = 4; 300 μg/ml, n = 22), or SDGRG (300 μg/ml, n = 8), detected by ELISA. b Release of LDH from cardiomyocytes incubated with PBS (control, n = 38), GRGDS (300 μg/ml, n = 22), or SDGRG (300 μg/ml, n = 8). (*p < 0.001 vs control, #p < 0.001 vs GRGDS 100, $p < 0.001 vs GRGDS 200, ‡p < 0.001 vs GRGDS 300, and §p < 0.001 vs SDGRG 300)Fig. 2Western blots of intact cTnI (29 kDa) and cTnI degradation products present in cell samples (left, 2.5× diluted) and medium samples (right, 10× concentrated) of cardiomyocytes incubated with PBS (control), GRGDS (300 μg/ml, a and b), SDGRG (300 μg/ml, c and d), or sodium azide (1 mmol/l, e and f) for 24 h
Effect of integrin stimulation on cell viability
Necrotic cell death was quantified by the release of LDH activity from cardiomyocytes into the medium (Fig. 1b). Cellular LDH activity of ten cultures was 0.47 ± 0.24 U per culture (100%). In control cardiomyocytes, ≈15% of total LDH was released in 24 h, which confirms previous findings of cellular necrosis in cardiomyocytes after 24 h in serum-free medium [25]. LDH release from integrin-stimulated cardiomyocytes (GRGDS, 300 μg/ml) equalled LDH release from control cardiomyocytes (Fig. 1b). Surprisingly, cardiomyocytes treated with SDGRG (300 μg/ml) for 24 h showed significantly less LDH release (9.5%) than control and GRGDS-treated cardiomyocytes (both p < 0.001).
Assessment of cell death using nuclear staining with propidium iodide showed a similar pattern. The percentage of necrotic cells in controls (7.2 ± 2.1%, n = 8) equalled that of GRGDS-treated cardiomyocytes (8.5 ± 2.2%, n = 8, p = N.S.). Again, the percentage of necrotic cells in SDGRG-treated cardiomyocytes was significantly lower (6.2 ± 1.9%, n = 8) compared with GRGDS-treated cardiomyocytes (p < 0.05), confirming the results of the LDH assay.
These results demonstrate that integrin stimulation by GRGDS participates in the release of cTnI from cardiomyocytes in the absence of necrosis.
Effect of integrin stimulation on MMP2 activity
In control experiments, we verified that recombinant active MMP2 is able to degrade purified cTnI in vitro in a number of fragments of 26, 14 and 8 kDa (Fig. 3). However, as we observed no cTnI degradation upon integrin stimulation (Fig. 2), we studied whether this was due to the lack of MMP2 activation during integrin stimulation, measuring the zymographic gelatinolytic activities of MMP2. In control cardiomyocytes, gelatinolytic MMP2 activities were detected in both cell and medium samples, indicating a baseline level of MMP2 in cultured cardiomyocytes (Fig. 4). Total MMP2 levels were higher in medium than in cells (≈90 vs ≈10% of total MMP2, respectively).
Fig. 3Western blot of intact cTnI (29 kDa) and cTnI degradation products after in vitro incubation of purified intact cTnI without and with active MMP2 and with active MMP2 in combination with a MMP2 inhibitor (o-phenanthroline) for 0, 60, and 120 minFig. 4Gelatinolytic activities of MMP2 isoforms in cell samples (left, 10× concentrated) and medium samples (right, unconcentrated) of cardiomyocytes incubated with PBS (control, n = 12), GRGDS (300 μg/ml, n ≥ 9), or SDGRG (300 μg/ml, n ≥ 3) for 24 h
In cells samples, several gelatinolytic activities of MMP2 were detected corresponding to a rodent-specific glycosylated MMP2 (75 kDa), proMMP2 (72 kDa, the major gelatinase activity), an active intermediate MMP2 (64 kDa), and active MMP2 (62 kDa; Fig. 4, left). GRGDS-treated cardiomyocytes demonstrated significantly higher levels of cellular proMMP2 (72 kDa) and active MMP2 (64 + 62 kDa) than control and SDGRG-treated cardiomyocytes (Fig. 4, left), whereas levels of glycosylated MMP2 (75 kDa) were unaffected by GRGDS. In medium samples (Fig. 4, right), several gelatinolytic activities of MMP2 were detected, corresponding to a rodent-specific glycosylated MMP2 (75 kDa), proMMP2 (72 kDa), and active MMP2 (62 kDa), respectively. GRGDS-induced integrin stimulation resulted in significantly higher medium levels of glycosylated MMP2 (75 kDa) and active MMP2 (62 kDa) compared to medium samples of control and SDGRG-treated cardiomyocytes (both p < 0.05; Fig. 4, right). Medium levels of proMMP2 (72 kDa) were unaffected by integrin stimulation.
Release of cTnI from necrotic cardiomyocytes
To compare the release of cTnI from viable cardiomyocytes during GRGDS-induced integrin stimulation with the release of cTnI upon necrotic cell death, cardiomyocyte cultures were treated with sodium azide (1 mmol/l) to induce necrotic cell death. After 24 h of azide-treatment, LDH release from cardiomyocytes was 73.4 ± 10.5% (n = 7), indicating extensive necrotic cell death. Release of intact cTnI from these necrotic cardiomyocytes was 32.7 ± 13.8% (n = 7). Notably, in contrast to the release of intact cTnI from integrin-stimulated cardiomyocytes (Fig. 2b), cardiomyocyte necrosis leads to the release of at least four cTnI degradation products with molecular weights of 26, 20, 17, and 12 kDa (Fig. 2f).
Discussion
The major findings of this study are that integrin stimulation in cardiomyocytes by GRGDS leads to (1) release of intact cTnI from viable cardiomyocytes in the absence of necrosis and (2) increased levels of intracellular and extracellular MMP2 activity that, however, does not result in degradation of cTnI into its fragments. This study therefore indicates that the release mechanism of cTnI from viable cardiomyocytes upon integrin stimulation differs from cTnI release from necrotic cardiomyocytes, which is associated with extensive cTnI degradation.
Elevated plasma cTnI levels are frequently found in pathological conditions in which irreversible myocardial cell injury is not a prominent aspect. In patients with heart failure, elevated plasma cTnI levels have been reported with normal plasma levels of CK-MB [2, 18]. Some studies have suggested that elevated serum levels of cTnI may be the result of myocardial strain [15, 20], as occurs during pressure or volume overload. Logeart et al. [15] studied 71 patients with heart failure and found in 19 patients elevated cTnI concentrations associated with LV remodeling and increased plasma brain natriuretic peptide levels. They postulated that increased LV wall strain may have led to the increased cTnI release, but necrotic cell death was not excluded in that study. In acute pulmonary embolism, several reports showed elevated cTnI levels [17, 21] possibly related to the presence of myocardial strain. In addition, Feng et al. [6] demonstrated that increased preload in isolated rat hearts was sufficient to cause release of cTnI as the result of myocardial stretch independent of ischemia.
Overload-induced stretch at the cardiomyocyte level is sensed by integrins, mechanotransducers molecules that link the extracellular matrix to the intracellular cytoskeleton [32]. In the present study, we showed that GRGDS-induced integrin stimulation of cardiomyocytes resulted in a threefold increased release of cTnI that occurred in the absence of necrosis, as cell viability remained unchanged by GRGDS treatment compared with control cardiomyocytes. Control cardiomyocytes demonstrated baseline levels of necrosis after 24 h, consistent with previous findings of cell death in cardiomyocytes after 24 h in serum-free medium [25]. Surprisingly, we found significantly less necrosis in SDGRG-treated cardiomyocytes than in control cardiomyocytes with both LDH assay and nuclear staining with propidium iodide. The mechanism responsible for this protective effect is unlikely to be related to integrin function but may be related to protection against necrosis exerted by certain proteins such as serum components.
Several studies have suggested that release of cTnI from viable cardiomyocytes, without lethal disruptions of the cardiomyocyte sarcolemma, does not involve intact cTnI (29 kDa) but rather proteolytic degradation products of cTnI formed by intracellular proteases as MMP2 and calpain-I [6, 7]. In the present study, we demonstrated that purified intact cTnI can indeed be degraded by active MMP2 in vitro. MMPs are a family of zinc-containing enzymes involved in degradation of the extracellular matrix during tissue remodeling [34]. However, MMP2 also acts intracellularly and may, therefore, be responsible for degradation of myofilament proteins including cTnI and myosin light chain-1 (MLC-1) [30, 39]. MMPs are synthesized by a variety of cells, including cardiomyocytes [4], in a latent form (proMMP) that is activated by either proteolytic cleavage or by conformational changes induced by cytokines, reactive oxygen species, and peroxynitrite [22, 35, 38]. Previously, Wang et al. [37] have demonstrated that mechanical stretch of neonatal cardiomyocytes induced expression and activation of MMP2. In addition, integrin stimulation by GRGDS has been reported to upregulate MMP2 expression in fibroblasts and certain tumor cell lines [31, 40], suggesting an important role for integrins in regulating MMP2 activity. In the present study, we showed that GRGDS-induced integrin stimulation of cultured cardiomyocytes was indeed associated with increased activity of intracellular proMMP2 and active MMP2 and increased release of active MMP2 into the medium. In cardiomyocytes incubated with SDGRG, neither cellular levels nor medium levels of proMMP2 or active MMP2 were altered compared to control cardiomyocytes, indicating that increased MMP2 activity is specific for GRGDS-induced integrin stimulation.
However, despite the increase of MMP2 activity during integrin stimulation and the ability of active MMP2 to degrade purified cTnI in vitro, integrin stimulation was not associated with cTnI degradation. If cTnI degradation had occurred, we would have been able to detect that in this system (Figs. 3 and 2f). The reason for the lack of cTnI degradation in the presence of GRGDS-induced MMP2 activation is unclear. The discrepancy between the in vitro degradation of purified cTnI by active MMP2 and the lack of cTnI degradation in GRGDS-treated cardiomyocytes may be explained by a difference in specific MMP2 activity. The MMP2 activity per nanogram cTnI in the study with purified cTnI was fourfold higher than that in cardiomyocytes treated with RGD. In addition, purified cTnI may be more susceptible to MMP2 cleavage than cTnI that is complexed to cTnT, cTnC, and tropomyosin. In cardiomyocytes, 93–97% of cTnI is complexed to cTnT, cTnC, and tropomyosin which may protect the MMP2 cleavage sites on the cTnI molecule.
The mechanism responsible for the release of intact cTnI from viable integrin stimulated cardiomyocytes may be leakage of free intact cTnI from the cytosolic pool. Several studies have demonstrated that mechanically induced transient disruptions (wounding) of the sarcolemma are a constitutive event in vivo [3, 10, 16, 23]. This mechanism is responsible for the release of proteins, such as myocyte-derived growth factors (fibroblast growth factors 1 and 2), which are released despite the lack of a classic signal peptide sequence normally associated with exocytotic secretion. These mechanically induced alterations in cardiomyocyte’s sarcolemmal permeability may similarly be involved in the release of cTnI from the cytosolic pool of cardiomyocytes in the absence of necrotic cell death. Further study is needed to address this issue.
Conclusions
The present study demonstrates that viable cardiomyocytes release cTnI as an intact protein by a stretch-related mechanism mediated by integrins. This finding may possibly explain why, in several pathological conditions, plasma cTnI levels are elevated in the absence of myocardial necrosis.

cardiomyocyte
stretch
cell death
mechanoreceptor
cell culture


What is new in otitis media?

The “wait and see” approach in acute otitis media (AOM), consisting of postponing the antibiotic administration for a few days, has been advocated mainly to counteract the increased bacterial resistance in respiratory infections. This approach is not justified in children less than 2 years of age and this for several reasons. First, AOM is an acute inflammation of the middle ear caused in about 70% of cases by bacteria. Redness and bulging of the tympanic membrane are characteristic findings in bacterial AOM. Second, AOM is associated with long-term dysfunction of the inflamed eustachian tube (ET), particularly in children less than 2 years of age. In this age group, the small calibre of the ET together with its horizontal direction result in impaired clearance, ventilation and protection of the middle ear. Third, recent prospective studies have shown poor long-term prognosis of AOM in children below 2 years with at least 50% of recurrences and persisting otitis media with effusion (OME) in about 35% 6 months after AOM. Viruses elicit AOM in about 30% of children. A prolonged course of AOM has been observed when bacterial and viral infections are combined because viral infection is also associated with ET dysfunction in young children. Bacterial and viral testing of the nasopharyngeal aspirate is an excellent tool both for initial treatment and recurrence of AOM. Antibiotic treatment of AOM is mandatory in children less than 2 years of age to decrease inflammation in the middle ear but also of the ET particularly during the first episode. The best choice is amoxicillin because of its superior penetration in the middle ear. Streptococci pneumoniae with intermediary bacterial resistance to penicillin are particularly associated with recurrent AOM. Therefore the dosage of amoxicillin should be 90 mg/kg per day in three doses. In recurrent AOM with β-lactamase-producing bacilli, amoxicillin should be associated with clavulanic acid at a dose of 6.4 mg/kg per day. The duration of the treatment is not established yet but 10 days is reasonable for a first episode of AOM. OME may be a precursor initiating AOM but also a complication thereof. OME needs a watchful waiting approach. When associated with deafness for 2–3 months in children over 2 years of age, an antibiotic should be given according to the results of the bacterial resistance in the nasopharyngeal aspirate. The high rate of complications of tympanostomy tube insertion outweighs the beneficial effect on hearing loss. The poor results of this procedure are due to the absence of effects on ET dysfunction. Pneumococcal vaccination has little beneficial effects on recurrent AOM and its use in infants needs further studies. Treatment with amoxicillin is indicated in all children younger than 2 years with a first episode of AOM presenting with redness and bulging of the tympanic membrane. Combined amoxicillin and clavulanic acid should be given in patients with β-lactamase-producing bacteria. The duration of treatment is estimated to be at least 10 days depending on the findings by pneumo-otoscopy and tympanometry. Bacterial and viral testing of the nasopharyngeal aspirate is highly recommended particularly in children in day care centres as well as for regular follow-up. The high recurrence rate is due to the long-lasting dysfunction of the eustachian tube and the immune immaturity of children less than 2 years of age.
Introduction
The number of publications relating to acute otitis media (AOM) and otitis media with effusion (OME) has been increasing markedly within the last 2 years [40–49]. It points to the difficulties in giving generally accepted guidelines for treatment. The confusion can be partially explained by the absence of uniform definitions and inhomogeneous groups of patients being included in clinical studies.
AOM is an acute inflammation of the middle ear, frequently elicited by viral infections and characterized by redness and bulging of the tympanum. Infections with bacteria can be identified in at least 70% of the cases by culture of the middle ear purulent fluid containing mainly polymorphonuclear cells [35].
OME is an effusion of the middle ear without symptoms of acute inflammations such as pain and fever, with frequently an amber or opaque aspect of the tympanum, the effusion containing mainly macrophages. Retraction of the tympanum with the light reflex still present may render the diagnosis difficult. Cultures for bacteria are positive in about 50% of cases whereas viruses have been identified in about 30% [35]. OME may be a precursor event initiating suppurate AOM but it can be the continuum of AOM as well. OME is the direct consequence of impaired middle ear ventilation.
Tympanometry (Fig. 1) shows a flat curve type b in both AOM and OME. In contrast, in OME associated with incomplete obstruction of the eustachian tube (ET) a curve c with a peak in the negative pressure zone is observed. Moreover, lower peak height and greater width in the negative pressure zone are frequent findings in OME [54].
Fig. 1The three main types of tympanograms showing: a a peak near atmospheric pressure expressed in daPa with stapedius reflex for different Hz frequencies, b a flat curve without stapedius reflex for 500, 2000, 4000 Hz pointing to presence of middle ear exudate: AOM or OME, c a lower peak in the negative pressure zone (−235 daPa) with an enlarged width pointing to OME with partial permeability of ET to air entrance and still a stapedius reflex
This overview summarizes the main new findings about AOM and OME which are in accordance with some basic well-known but sometimes forgotten data and facts, with particular emphasis on the vulnerability of young children below the age of 2 years.
Aetiopathogenesis
Role of the eustachian tube
The great importance of the first attack of AOM in young children lies in the subsequent long-lasting dysfunction of the ET. Indeed, the main functions of the ET are ventilation, protection and clearance of the middle ears (Fig. 2) [5, 35] and play a determining role in the recurrence of AOM. Ventilation of the middle ear occurs by each deglutition by action of the tensor velum palatinum muscle leading to air equilibration with the atmospheric pressure. By obstruction of the ET, a negative pressure develops inside the middle ear resulting in effusion and aspiration of nasopharyngeal secretions. Poor ventilation leads to decreased PO2 which results in decreased bactericide power of polymorphonuclear cells. Impaired clearance results in proliferation not only of aerobic but also anaerobic bacteria in the middle ear. Conversely, reflux otitis occurs with decreased compliance of the ET due to abnormal flaccidity.
Fig. 2Three physiologic functions of the eustachian tube in relation to the middle ear. NP nasopharynx, ET eustachian tube, TVP tensor velum palatinum muscle, ME middle ear, MAST mastoid, TM tympanic membrane, EC external canal
ET dysfunction is a major problem in the young child: the small calibre of the ET as well as its horizontal direction [5] (Figs. 3 and 4) are responsible for both the high incidence of AOM and the frequent relapses by each viral infection. This explains the poor long-term prognosis evidenced in a prospective study comprising 210 AOM patients of less than 2 years of age by Damoiseaux et al. [16]. At least 50% of the patients had recurrences of AOM and persistence of OME was observed in 47% of them after 3 months and in 35% after 6 months. Yet, only 50% of these patients were treated with amoxicillin. Unfortunately, the authors do not mention either the delay of treatment or the dosage of the antibiotic.
Fig. 3The difference in the angle of the eustachian tube between infants and adults [5]Fig. 4The tympanic membrane forms the lateral wall of the box-shaped middle ear. The function of the eustachian tube is to equilibrate middle ear pressure with that in the nasopharynx. Bacteria and viruses resident in the nasopharynx may reach the middle ear during pressure equilibration. One-third of the middle ear mucosa and the entire eustachian tube are lined with mucociliary epithelium to transport bacteria from the middle ear back to the nasopharynx. Air from the middle ear enters the mastoid air cells by way of the aditus
In other words, the first episode of AOM determines the evolution and the recurrences because of the associated acute inflammation involving also the ET.
Role of viruses and bacteria
Viruses
Most children are infected with respiratory syncytial virus (RSV) in their first year of life. A prospective study [2] in 42 children aged 2–24 months with bronchiolitis showed that 26 of them had AOM at entry or within 10 days and an additional 10 developed OME; only 6 patients remained free of both AOM and OME during a 3-week observation period. These findings were confirmed in a recent study showing that in patients with persistent RSV antigen in the middle ear effusion, 31% relapsed despite a favourable outcome of the first AOM episode [50].
The association of AOM with several viral infections has been largely documented. The prevalence of respiratory viruses in the middle ear fluid of 456 children, aged 7 months to 7 years, with AOM was 41% [21]. RSV was the most frequent virus isolated followed by parainfluenza, influenza, enteroviruses and adenoviruses. These findings have been confirmed by others [18, 32, 33, 41, 50] and rhinovirus, coronavirus and metapneumovirus should be added to the above-mentioned list [53]. Even the measles virus has recently been isolated from the middle ear fluid in two patients [64].
Bacteria
In 70% of patients with AOM, bacteria can be found by culturing the middle ear fluid [35]. The most frequent species isolated are Haemophilus influenzae and Streptococcus pneumoniae [27]. Nasopharyngeal aspirate culture may give valuable information on the bacteria involved in AOM [28, 51, 55, 58]. The presence of conjunctivitis points to Haemophilus influenzae infection [4].
Heikkinen et al. found Streptococcus pneumoniae in 25% of their patients, Haemophilus influenzae in 23% and Moraxella catarrhalis in 15% [21]. It has been demonstrated that recurrences of AOM are associated with positive bacterial culture in the nasopharynx [23], even at the end of the antibiotic treatment [28] and in most cases the pathogen was Streptococcus pneumoniae [58]. However, recurrence more than 14 days after an initial AOM episode is mostly due to a new infection, i.e. not a true relapse [27]. In a most recent prospective study on purulent meningitis [11], the association of AOM was found in half of the patients [14].
Bacteria and viruses
Bulut et al. performed both bacterial and viral testing in 120 children with AOM [8]. These patients, aged between 6 months and 12 years, did not receive an antibiotic for 2 weeks prior to the study. A positive bacterial culture was obtained in 54% of the children and respiratory viruses were identified in 32% of them; combined viral and bacterial infection was demonstrated in 12% of the samples.
Persistent otitis was documented in about 50% of the children with combined viral and bacterial infection in a prospective study of 271 children because viral infections contribute to the ET dysfunction [12].
Other risk factors
The immaturity of the immune system of young children makes them incapable of killing encapsulated bacteria. This, along with the ET dysfunction, explains the long-lasting course of AOM as well as the high recurrence rate.
This is particularly important for infants attending day care centres [37, 38]. It is sometimes necessary to withdraw these infants from these institutions during the winter to avoid recurrences. Additional risk factors are passive smoking, young siblings going to school and siblings with previous AOM episodes.
These factors were identified in a prospective study on 2,253 children aged 2 months to 2 years published in 1997 [37]. The proportion of patients developing at least one episode of OME was 79% at 12 months and 91% at 24 months. Breast feeding and exposure to tobacco smoke contributed little to the outcome, but a lower socioeconomic status and repeated exposure to other children at home or in day care centres were the most important risk factors.
Children with cleft palate are particularly prone to otitis and should be treated by early tympanostomy tube insertion [60].
Complications: mastoiditis
Mastoiditis should be treated by antibiotics according to the bacterial resistance and by temporarily inserting tympanostomy tubes to improve drainage [65] because of the narrow calibre of the aditus ad antrum. [22] (Fig. 4).
Treatment
General considerations
A “wait and see” approach has been advocated in the treatment of AOM in children mainly because of the increased antimicrobial resistance of bacteria causing respiratory infections [13]. Indeed, a meta-analysis of 5,400 children, aged 6 months to 18 years, and collected from 33 randomized trials concluded that there was a significant but modest impact of antibiotics on the primary control of AOM [45]. Similar results emerged from a subsequent study published by Damoiseaux et al. in 2000 [15]. This randomized double-blind prospective trial carried out by general practitioners in 240 children, aged 6 months to 2 years, showed that only 7 and maybe 8 of them needed antibiotics. Both studies, however, suffer from major flaws; the absence of long-term data, in particular the recurrence rate of AOM, and OME not being considered an end point. Moreover, the former study included patients with a very large age range and therefore blunted the effect of the critical age group of the patients below the age of 2 years. Indeed, over 70% of AOM patients present their first attack during the first year of life and over 90% within 2 years of age [35]. The visualisation of the tympanum in this age group is not always easy especially when cerumen obstructs the narrow external ear channel. Yet, as already mentioned above, bulging and redness of the tympanic membrane are important diagnostic signs of bacterial AOM [27, 39].
Treatment of AOM
The crucial role of the first attack of AOM makes the immediate administration of antibiotics mandatory in all patients with AOM with redness and a bulging tympanum (Table 1) [26]. A “wait and see” approach can only increase the inflammation and hence the ET dysfunction. The choice of the antibiotic must take into account the penetration in the middle ear and the type of bacteria involved. The penetration for amoxicillin—about 40% of the blood concentration—is much higher than for cephalosporins (about 20%) and macrolides (about 10%) but lower than for cotrimoxazole (60%) [31]. The penetration of amoxicillin is not changed by the addition of clavulanate to amoxicillin [10, 52].
Table 1Guidelines for treatment of AOMEpisodeSigns or symptomsTreatment or approachChildren <2 years of ageFirst episodeAmoxicillin 90 mg/kg per day for at least 10 days according to the bacterial resistanceWeekly follow-up by tympanometry and pneumo-otoscopyRecurrent episodeWith redness and bulging tympanumAmoxicillin or amoxicillin 90 mg/kg per day + clavulanic acid 6.4 mg/kg per day according to bacterial resistanceWithout bulging“Wait and see” approachChildren >2 years of ageWith redness and bulging tympanum or otorrhoeaSame treatment as “recurrent episode”Without evident bulging tympanum“Wait and see” approach
Because of the increased incidence of Streptococcus pneumoniae with intermediary resistance [57, 61], the dosage of amoxicillin should be doubled, i.e. 90 mg/kg per day given in three administrations as recommended by the Centers for Disease Control and Prevention [3].
Haemophilus influenzae is more likely to become β-lactamase positive after previous antibiotic administration [9]. Therefore, the addition of clavulanate—at a dose of 6.4 mg/kg per day—to amoxicillin makes sense. Most Moraxella catarrhalis strains are nowadays also β-lactamase positive [42].
The duration of the antibiotic treatment is a matter of discussion. For children less than 2 years of age, a 10-day course is reasonable. Pneumo-otoscopy and tympanometry can help to appreciate the permeability of the ET. Careful follow-up is necessary.
Nasopharyngeal aspirate cultures may give valuable results and are highly recommended in children remaining in day care centres or with a history of previous AOM episodes [28, 51].
For children more than 2 years of age the previously watchful guidelines can be maintained but some flexibility is recommended [22, 36].
Treatment of OME: tympanostomy tubes?
The approach to OME must take into account the natural history of spontaneous healing and the main complication which is temporary hearing impairment [1]. The absence of spontaneous healing after 6 weeks to 2 months in children over 2 years of age is an indication for a 10-day antibiotic course according to the results of the nasopharyngeal aspirate cultures. It is our experience that this approach has several advantages. First, the effect even temporarily on the hearing defect is frequently successful. Second, it decreases the recurrence of AOM by improving the ET functions and third, it allows postponement or avoidance of tympanostomy tube insertion, because improved hearing points to improved permeability of the ET.
The delay of 6 weeks or 2 months before treatment of OME (Table 2) is based on the duration of OME. Indeed, in a prospective study by Renko et al. [43] of 90 children with AOM, 0.6–7 years of age, treated with amoxicillin or cefuroxime axetil for 10 days, the mean duration of OME was 10.2 days (range: 1–58 days); only 10 of 90 still had OME after 29–43 days.
Table 2Guidelines for treatment of OME. SMZ sulfamethoxazole, TMP trimethoprimIndicationApproachTreatmentWithout deafness“Watchful waiting” approachWith deafnessAfter 6 weeks-2 months in children ≥2 yearsAmoxicillin 90 mg/kg per day or amoxicillin 90 mg/kg per day + clavulanic acid 6.4 mg/kg per day for 10 days according to the results of the bacterial resistanceNo swimmingRecurrent OME or OME of long durationAzithromycin 10 mg/kg per day, 3 days/week for 4 weeksCotrimoxazole 30 mg SMZ + 6 mg TMP/kg per day according to the results of the bacterial resistanceAdenoidectomy in children ≥3 years of age with nasopharyngeal obstruction and recurrent adenoiditisNo swimming
In a long-term prospective study in 6,350 healthy infants treated according to specified guidelines, Paradise et al. [38] observed bilateral OME in only 5.6% of the 161 children who had not received tympanostomy tubes at the age of 4.
The indication to insert tubes is equivocal. The large prospective studies on 3,686 infants and young children by the Paradise group have shown the absence of or negligible effects of tympanostomies on developmental outcomes at 3–4 years [34, 37]. On the other hand, insertion of tympanostomy tubes during the first 3 years of life is associated with complications in at least 80% of the patients [63]. Tympanostomy can be a source of bacterial infections [24] and of tympanosclerosis.
Extensive reviews by Kubba et al. [25], Rosenfeld et al. [46], Rovers et al. [48] and the recent Cochrane Database by Lous et al. [30] concluded that there was only a small benefit of the conventional ventilation tube. The main reason for the poor results of tympanostomy tubes is the absence of effects on the ET dysfunction. It has never been demonstrated that tympanostomy tubes improve the functions of the ET.
The recommendations by the American Academy of Pediatrics [1] are to insert tubes in conditions with persisting hearing loss of more than 40 dB particularly with posterosuperior retraction pockets, ossicular erosions and adhesive atelectasis because of the increase of structural damage with effusion duration.
Watchful waiting for 3–6 months in the winter period seems advisable in children with mild hearing deficit.
Adenoidectomy should be considered in children ≥3 years of age with nasopharyngeal obstruction and recurrent adenoiditis [45].
Vaccination
The effects of pneumococcal vaccination on recurrent AOM have been studied extensively. In a randomized controlled trial by Brouwer et al. [6] in 383 children, aged 1–7 years, with recurrent AOM, the effects in 190 children treated with heptavalent pneumococcal vaccine were compared with 193 children receiving hepatitis A or B vaccines. The study concluded that there was no beneficial effect. A similar conclusion was drawn from a study by Van Kempen et al. [62] in 74 children aged 1–7 years and in the Cochrane Database by Straetemans et al. [56].
A shift of the causative pathogen has been observed from Streptococcus pneumoniae to Haemophilus influenzae or to other strains of pneumococci than the ones included in the vaccine [20, 59]. A slight reduction of the incidence of AOM of 6–8% was observed in the USA [7].
Infants and prematures
A high incidence of OME has been observed in neonatal intensive care units due to the nasally placed tubes for ventilatory assistance. It has been suggested that immune immaturity with impaired neuromotor function predisposes to this complication [17]. Vaccination with the heptavalent pneumococcal vaccine has been advocated in premature babies and neonates [19], but this study does not provide information on the actual AOM prevalence in a control group receiving only hepatitis B vaccine. It should be remembered that meconium aspiration into the entry of the middle ear is associated with increased risk of AOM and therefore early nasopharyngeal suction is mandatory [29].

acute otitis media
aom
otitis media with effusion
ome
tympanostomy tube insertion
vaccination
guidelines for treatment
mastoiditis
eustachian tube dysfunction
otitis media in infants


Maximum tumor diameter is not an independent prognostic factor in high-risk localized prostate cancer

Objectives Previous studies suggest that maximum tumor diameter (MTD) is a predictor of recurrence in prostate cancer (PC). This study investigates the prognostic value of MTD for biochemical recurrence (BCR) in patients with PC, after radical prostatectomy (RP), with emphasis on high-risk localized prostate cancer.
Introduction
The characteristics mostly used to predict progression of prostate cancer (PC), after radical prostatectomy (RP), are Gleason score, pre-operative PSA level, pathological staging, positive surgical margins, and tumor volume (TV). [1] The first three are also used to define high-risk localized PC [2]. TV in the RP specimen is also clearly associated with prognosis. However, whether it is an independent prognostic factor, both for the total group and for the high-risk group, still remains controversial [3].
Epstein et al. [4] stated that, although TV is a predictor of progression, it did not provide additional information over Gleason score and pathological stage. This was also found in a study by Salomon et al. [5] where only Gleason score and pathological stage were independent factors to predict progression after RP. If these parameters were known, TV did not provide any additional information. This is in contrast with Stamey et al. [6] who found that TV is an independent prognostic parameter for PC progression. These findings were also observed in other studies [7–9]. None of these studies, however, specifically addressed the high-risk subgroup. An explanation of these conflicting results might be that TV estimations can be done with the help of several techniques, as Humphrey and Volmer [10] described earlier. Moreover, these techniques are rather complex and time-consuming. Therefore, maximum tumor diameter (MTD) has been suggested as a fast and easy proxy for TV. Renshaw et al. [11] noted that simple measurements of the largest diameter of the largest tumor from glass slides is a predictor of PSA failure. This was supported by Eichelberger et al. [12] who showed that MTD correlates with TV and other potential prognostic factors for clinical outcome. However, these studies had either a small number of patients with a relatively short follow-up (57 patients, median follow-up 27.2 months) or did not investigate the correlation between MTD and biochemical recurrence (BCR).
Therefore, in this present study, with a large cohort of 542 RP specimens, and a median follow-up of 39.5 months, we studied the prognostic value of MTD, as a surrogate for tumor volume, for BCR in patients with PC, with special attention to the high-risk group.
Materials and methods
Between 1992 and 2005, 617 patients were treated with RP for clinically localized PC at our institute. Of these patients, 75 were excluded from analyses because of HIFU treatment, hormonal pre-treatment or irradiation before RP. Of the remaining 542 patients, five had incomplete follow-up data. Charts were examined retrospectively for clinical follow-up data and risk group classification. These risk groups were classified according to D’Amico et al. [2]. High risk was defined as stage ≥T2c or PSA level >20 ng/ml or Gleason score ≥8, and we formed one group of the low and intermediate risk group (stage < T2c, PSA level ≤ 20 ng/ml and Gleason score ≤ 7).
All RP specimens were fixed overnight, inked, and cut into serial, transverse, 4 mm thick slices according to a standard protocol by Ruijter et al. [13]. In brief, the apical and basal margins were amputated to a thickness of 4 mm and sectioned parasagittally at 4 mm intervals. The seminal vesicles were sectioned parallel and entirely submitted. The prostate was then sectioned perpendicularly to the long axis (apical to basal) of the gland along the posterior rectal surface at 4-mm intervals. Tumor was outlined on the microscopic glass slides and schematically drawn on the macroscopically photographed transverse sections in order to be able to reconstruct tumor extension and tumor multifocallity. Detailed pathological analysis, including number of tumors, total volume of all tumors, MTD, pathological staging, Gleason score, extracapsular extension and positive margins, was performed by a single pathologist (CAHK). Tumors were staged using the American Joint Committee on Cancer (AJCC) TNM staging criteria of 2002 [14]. MTD was defined as the largest diameter of the largest tumor. MTD was determined by marking both ends of the tumor and measuring the distance at the glass slide directly. If the tumor was present on consecutive glass slides, thickness of the slices of the concerning sections was summed. This reconstructed diameter was considered as MTD if it was larger than the diameter, and was visualized directly on the glass slides.
PSA values were obtained before surgery and at every follow-up point. BCR after RP was defined as two subsequent PSA levels above 0.10 ng/ml among patients who reached non-measurable levels after RP.
Statistical analysis
Baseline characteristics are summarized with median and interquartile ranges (IQR). Associations between MTD and clinical or pathological characteristics were examined by univariate regression models. Kaplan–Meier curves were used to assess the risk of BCR. Proportional hazards multivariable regression models were composed to determine prognostic factors for BCR. SPSS version 12.0.1 for Windows was used for all statistical analysis.
Results
Patient and tumor characteristics are summarized in Table 1. The age of the patients at time of RP was 62.7 years (IQR 58.4–66.5 years) and they had a median Gleason score of 6. In the specimens, a median MTD of 24.0 mm (IQR 16.0–32.0 mm) was found. Patients were followed for a median period of 39.5 months (IQR 17.1–67.3 months). Overall, 114 (21%) patients developed BCR after RP. The median time to PSA recurrence was 20.3 months (IQR 10.3–46.9). The overall 5-year Kaplan–Meier risk of BCR was 25% (95% CI = 20.4–29.6) for the whole group. For the high-risk group and low/intermediate risk, these were 27.4 (95% CI = 22.2–32.6) and 10% (95% CI = 2.2–17.8), respectively. Totally, 432 of the 542 patients fulfilled the criteria for high-risk localized PC, 72 patients were low/intermediate risk, and 38 patients never reached non-measurable PSA levels.
Table 1Patient and pathological characteristics (n = 542)MedianIQRAge (years)62.758.4–66.5Follow-up period (months)39.517.1–67.3Pre-operative PSA (ng/ml)8.05.5–13.0Gleason score65–7Number of tumors (n)21–3Index tumor volume (cm3)1.60.6–3.4Total tumor volume (cm3)2.00.8–3.9Maximal tumor diameter (mm)24.016.0–32.0Data are presented as median and interquartile ranges (IQR)
Associations between MTD and pathological characteristics are summarized in Table 2. Overall we found 359 organ-confined (T2) tumors, 116 extracapsular extended tumors (T3a), and 52 tumors invaded into seminal vesicles (T3b). Half of all the patients (n = 271) had positive surgical margins and eight patients had lymph node involvement. Pre-operative PSA levels ranged from 0.1–87.2 ng/ml (median 8.0 ng/ml). Kruskall–Wallis tests or Mann–Whitney U test revealed that MTD was significantly associated with pathological stage (P < 0.001), Gleason score (P < 0.001), margin status (P < 0.001), number of tumors (P < 0.001) and pre-operative PSA levels (P < 0.001). Positive surgical margins were associated with a larger MTD compared to negative surgical margins. Single tumors were also found to have a larger MTD than multiple tumors. Extracapsular extension (T3a) and invasion to seminal vesicles (T3b) were both associated with larger maximal tumor diameters compared to those of organ-confined (T2) tumors. MTD in the high-risk group was significantly higher then in the low/intermediate risk group (median 24.5 vs.14.0 mm, P < 0.001). We also calculated the correlation coefficient for MTD and all other factors. We found that MTD was significantly correlated to index TV (=largest tumor) (r = 0.60, P < 0.001) and total TV (r = 0.61, P < 0.001) in the total group. In the high-risk group, these correlations were weaker. In addition, significant correlations with MTD were found for Gleason score (r = 0.17, P < 0.001), the number of tumors (r = −0.21, P < 0.001) and pre-operative PSA levels (r = 0.38, P < 0.001).
Table 2Associations between MTD and pathological characteristicsNo. of patientsMaximum tumor diameterMeanMedianRangeP-value*Pathological stagea (2002) T235921.020.01–60<0.001 T3a11630.828.012–60 T3b5231.131.03–55Gleason scorea ≤630122.521.01–65<0.001 3 + 413626.225.08–60 4 + 32225.024.03–49 ≥88028.828.04–60Margin statusb Negative27020.520.01–52<0.001 Positive27128.528.03–65Number of tumorsb 124127.326.01–65<0.001 ≥130122.322.01–50Pre-operative PSAa <45617.415.03–46<0.001 4–1028923.624.01–55 10–2012324.524.02–55 >207034.435.510–65Risk groupsb High 43225.824.51–65<0.001 Low/intermediate7214.314.01–36* P-values were assessed by aKruskal–Wallis test or bMann–Whitney U test
Univariable analyses of MTD as a continuous variable revealed that it is weakly associated with risk of BCR (HR = 1.02 per mm increase, 95% CI = 1.00–1.04, P = 0.024). In the high-risk group, this association was lost (HR = 1.01 per mm increase, 95%CI = 0.99–1.03, P = 0.18).
In the total group TV, pre-operative PSA levels, pathological stage, and Gleason score were associated with risk of BCR, as presented in Table 3. Proportional hazards multivariable regression models were composed to determine significant independent prognostic factors (Table 3). Positive surgical margins, higher Gleason score, advanced pathological stage, and multiple tumors were the main prognostic factors for BCR. Neither MTD nor tumor volume turned out to be significant in either group.
Table 3Univariable and multivariable analysis of clinical and pathological characteristics associated with time to BCRCovariatesUnivariableMultivariableHR95% CIAHR95% CIPre-operative PSA1.021.01–1.04*Pathological stage pT21.01.0 pT3a2.121.39–3.23*1.260.81–1.97# pT3b3.902.33–6.53*1.791.02–3.13*Gleason score ≤61.01.0 3 + 42.641.64–4.25*1.971.20–3.23* 4 + 37.273.18–16.63*5.712.47–13.20* ≥85.683.60–8.97*3.572.17–5.89*Extracapsular extension2.711.87–3.94*Invasion seminal vesicle3.071.94–4.87*Margin status4.643.02–7.12*3.752.36–5.96*Number of tumors1.020.88–1.17#1.181.02–1.36*Maximal tumor diameter1.020.99–1.04#Index tumor volume1.041.02–1.07*Total tumor volume1.041.02–1.07*HR hazard ratio, CI confidence interval, AHR adjusted hazard ratio* P < 0.05, # P > 0.05
Discussion
Prediction of outcome after RP in patients with localized PC is important to tailor follow up, and for example, to consider adjuvant therapy in patients at high risk for recurrence. Several studies have assessed the value of morphological and clinical variables in this respect. Tumor stage, Gleason score, and pre-operative PSA levels are obvious parameters, and also used to define patients at high-risk for recurrence [2]. MTD, as easy to determine surrogate for tumor volume [12], is another potential variable considered as a potential predictor of BCR. In our study of 542 patients, we found that MTD correlates with index TV, total TV, pathological stage, Gleason score, number of tumors, and pre-operative PSA levels irrespective of risk classification. However, MTD was not an independent predictor of BCR in the total or in the high-risk subgroup. For the whole group, these results are in contrast with previously reported findings.
One of the first studies addressing the association of MTD and BCR was by Renshaw et al. [3]. In this study, with 57 patients undergoing RP, it was found that MTD was strongly correlated with total TV. In this small series, a high MTD was associated with a slight increased risk for BCR (HR 1.12) that was marginally significant. Their subsequent study was a larger cohort of 434 patients. The median MTD was 13 mm. The median follow-up was 12.9 months and 27.2% of patients had PSA failure. This study confirmed that MTD is an independent predictor of BCR, even after adjustment for other risk factors [11].
Their next study by Dvorak et al. [15] had an even larger cohort (781 patients) and longer median follow-up (64.8 months). MTD remained the same with a median of 13 mm, and BCR in 31% of the patients. Again they found that MTD was significantly associated with time to BCR (HR = 1.04, P = 0.004) adjusted for pre-op PSA, Gleason score, and pathological stage. But when surgical margin status was added, MTD lost statistical significance (P = 0.07). One important feature of these studies is that the RP specimens were not uniformly processed. Therefore, we have to be taking into account the variation in evaluation of the prognostic factors. Another difference with our results is that the median MTD in these three studies (13 mm) are less than in our study (24 mm). This might be explained by the way we determine our MTD. Renshaw et al. limited their measurements to the single focus of tumor that could be demonstrated on one slide (slice-MTD). We, on the other hand, take the largest diameter of the largest tumor after reconstruction (real-MTD). To see if we could explain this difference, we also measured the slice-MTD in a part of our cohort. In 250 consecutive cases from 2000 to 2005, we found a median slice-MTD of 19 mm and a median real-MTD of 24 mm. This strongly suggests that part of the difference in MTD is caused by the different way MTD is measured. The difference that still exists could be explained by the fact that Renshaw et al. used partially submitted prostates, thus missing part of the prostates for evaluation. The only study where they did use uniformly processed specimens is by Eichelberger et al., but this study has a very short follow-up. They analyzed the prognostic value of MTD in a cohort of 364 patients treated with RP, and found a mean MTD of 17.3 mm and biochemical recurrence in 12% of the patients [16]. In this study with a median follow-up of only 12 months, they found that MTD was a significant predictor of BCR, adjusted for Gleason score and surgical margin status (HR = 1.70, P = 0.011). In our study we found a median time to BCR of 20.3 months, hence a follow-up of 12 months seems not long enough to investigate BCR.
Another striking difference is the percentage of positive surgical margins. In our study this is relatively high (50%) compared to other studies (Renshaw et al. 31 and 26%, Eichelberger et al. 23%, Dvorak et al. 27%). Our median BCR rate (21% after 39.5 months), however, was comparable to the median of these studies [Renshaw 27% (22.5 months), Eichelberger 12% (12 months), Dvorak 31% (64.8 months)]. An explanation for the high number of positive surgical margins, next to surgical techniques, is that by evaluating only partially included prostates you miss positive surgical margins.
The difference between our study and studies like Eichelberger et al, in which the entire prostate was histologically examined using whole mount sections, are less easily explained, but here the interval at which the prostates were sliced is higher (mean 7.8 mm according to the used protocol described by Qian et al. [17], personal communication). From this, we can conclude that MTD is not an independent prognostic marker if surgical resection margins are accurately documented.
Finally, none of the mentioned studies separately looked at the group where prediction might be of most importance, the high-risk group. As clearly indicated, in this group, neither MTD nor TV is of any significant value.
In all, we do not advice to use MTD as a prognostic factor for BCR, since after proper follow-up and with adequate pathological techniques it does not add anything to other readily available prognostic factors. Moreover, in the high-risk group, TV is not an independent prognostic factor at all.
Conclusions
The results of our study suggest that MTD is positively correlated to total TV, as can be expected. MTD was weakly associated with risk of BCR and this association was lost in the high-risk group. If adjusted for positive surgical margins, higher Gleason score, advanced pathological stage, and multiple tumors, MTD did not provide additional information.

localized
prostate
prostatectomy
prognosis
high risk