A survey of failed post-retained restorations

Survival of endodontically treated, post-restored teeth depends on a multitude of factors, all of which are practically impossible to include in a randomized, controlled clinical study. The purpose of this survey was to characterize and analyze reported failures of post-retained restorations to identify factors critical to failure and to type of failure. A questionnaire was mailed to private practitioners in Denmark with a request to complete the questionnaire whenever a patient presented with a failed post-retained restoration. Information was gathered on factors related to the patient, the tooth, the restorative materials, and the techniques. Two-hundred and sixty questionnaires were collected from 171 practitioners over a 3-year period. Functioning time until failure varied between 3 months and 38 years. Mean survival time until failure was 11 years. Of the failed restorations, 61% had functioned for 10 years or less. Fracture of the tooth was the most common type of failure reported, followed by loosening of the post and fracture of the post. Tapered posts implied an increased risk of tooth fracture compared to loosening or fracture of the post, and the relative risk of tooth fracture increased with the functioning time until failure. Fracture of the post was more common among male than female patients. On the basis of this survey of failed post-retained restorations, it was concluded that tapered posts were associated with a higher risk of tooth fracture than were parallel-sided posts.
Introduction
Endodontically treated teeth have been reported to have shorter survival time than vital teeth [14, 20, 29, 33, 35]. This is believed to reflect that endodontically treated teeth are often structurally compromised because of a series of events: caries or trauma, endodontic therapy, and preparation for restorative procedures [12, 19, 32, 34, 40, 41, 47–49]. Thus, to minimize the negative influence on longevity, the restorative procedure of endodontically treated teeth calls for careful consideration of treatment alternatives.
In the case of minimal-remaining tooth substance, the retention of a crown has traditionally been provided by a core and the retention of the core by a post. Two fundamentally different techniques find widespread use for the construction of a post and core: (1) a post and core cast as a single unit and (2) a prefabricated post upon which a core is directly produced in, e.g., amalgam or resin composite. The first prefabricated posts on the market were made of stainless steel or gold-plated brass. Later, posts of titanium alloys became popular, and recent developments have introduced posts made of fiber-reinforced resin composite or ceramic. Simultaneously, adhesive dentistry, offering reliable bonding to tooth substance and to restorative materials via various pretreatments, has markedly progressed to widen the possibilities also for post and core treatment.
As mentioned, the post serves to retain the restoration. However, the post should also serve to protect the remaining tooth structure. These two functions of endodontic posts may be evaluated by determining the retentive ability of the posts and the fracture resistance of endodontically treated teeth provided with post-retained restorations. The latter may also be indirectly determined by analysis of the stress-distributing characteristics of the posts. Post-related factors that have been found to exert influence on retention of posts and protection of tooth structure include: shape, length, diameter, surface design, and stiffness of the post as well as the type of luting cement used.
As regard to post shape, parallel-sided posts provide better retention and less stress formation and increased fracture resistance than do tapered posts [2, 5, 18, 28, 37, 38, 42, 46]. An increase in post length has a similar, positive effect [2, 5, 7, 8, 17, 42]. However, some restrictions apply to post length. First, the apical seal of the endodontic restoration must be ensured through maintenance of 5–6 mm of apical gutta-percha [1, 24, 53]. Second, dentin in the apical third of teeth is often very thin. This not only increases the risk of parietal perforation, but it also weakens the tooth to a relatively high degree, thereby increasing the risk of root fracture. Little evidence is available as regard to the influence of post diameter. Consensus regarding retention seems to be that an increase in post diameter cannot be relied on as a measure to improve retention [5]. A study based on finite element analysis found that stress formation in the dentin decreased when post diameter was increased [2]. Conversely, another study showed that thinner posts provided increased resistance to fracture [48]. A consensus regarding fracture resistance seems to be that the root canal should not be uncritically enlarged, as decreasing the bulk of dentin weakens the tooth and reduces the resistance to fracture [5, 12, 30]. As regard to surface design, serrated posts provide better retention than smooth-sided posts, and threaded posts provide even better retention [5, 7, 37]. However, the superior retention of threaded posts, obtained by engaging in dentin, simultaneously is accompanied by increased stress formation within the root [36, 43] and thus by an increased risk of root fracture [5, 10, 12, 43]. Post stiffness, which is a combination of post diameter and elastic modulus of the post material, is another important characteristic, but an unsettled discussion prevails as to optimal stiffness. On the one hand, insufficient stiffness will allow excessive distortion of the restoration at the margins during function, leading to breakdown of cement and risk of secondary caries. Moreover, a decrease in the elastic modulus of the post material has been found to increase stress formation in the root and to decrease fracture strength of the restored tooth [2, 23, 38, 39]. Thus, low-modulus posts fail sooner or at lower stress values than do high-modulus posts. On the other hand, several studies have found that low-modulus posts display failures that cause little damage to the remaining tooth structure (loss of marginal seal, loss of retention, core fracture, and post fracture), while high-modulus posts are associated with a higher incidence of root fractures when they finally fail; that is, they cause more damage to the remaining tooth structure and often result in the extraction of the tooth involved [22, 23, 39, 47]. Retention of posts in the root canal is primarily sought through the use of a luting agent. Zinc phosphate cement has been the luting agent of choice for many years. The progress within adhesive dentistry and the advent of ceramic- and fiber-reinforced resin composite posts have prompted questions of the usefulness of resin cement for the luting of posts. Zinc phosphate cement provides retention by mechanical interlocking in irregularities in the surface of the root canal on one side and in the surface of the post on the other. After appropriate pretreatment of the respective surfaces, resin cements provide retention not only by mechanical interlocking but also by micromechanical and chemical adhesion. In vitro studies have found improved retention, less and more favorable stress formation, and improved fracture resistance with resin cements than with zinc phosphate cement [2, 6, 25, 31, 51].
Other factors from those related to the post are of importance for success of post-retained restorations. Thus, researchers agree that the extent of remaining tooth structure is a key issue for fracture prognosis [9, 12, 30, 42, 47]. Another paramount factor is the ferrule effect: Teeth that allow preparation of a 1.5–2-mm ferrule show superior fracture resistance [44, 45, 54].
The foregoing has shown that numerous factors influence the longevity of endodontically treated teeth provided with post-retained restorations and that it would be practically impossible to carry out a randomized controlled clinical trial that includes all relevant variables. Previous studies have shown annual failure rates related to post and core treatment of up to 5% [4, 9, 11, 15, 41, 46]. The purpose of this survey of failed post-retained restorations was to analyze reported failures in an attempt to identify factors that are critical to failure and to type of failure.
Materials and methods
A questionnaire was developed and piloted, and in June 2000, it was posted, in five copies, to the members of the Danish Dental Association who worked in private practice (3,444 practitioners). The practitioners were asked to complete the questionnaire in case a patient presented with a failed post-retained restoration. Questions were asked about age and gender of the patient, tooth involved, number of posts, type, design, length, and diameter of the post(s), type of core, type of luting agent, type of restoration, height of any ferrule, degree of tooth destruction, functioning time until failure, and type of failure.
After 3 years, 260 questionnaires had been received, and the collection of questionnaires was terminated. Data were transferred to a computer and analyzed by use of the Statistical Analyzing System (SAS). Basic descriptive statistics including frequency distributions were performed. Selected data were analyzed by chi-squared tests and Fischer’s exact probability tests (α = 0.05). Cross tabulations were carried out to further characterize the data and to identify codeterminants of functioning time until failure and codeterminants of type of failure.
Results
The 260 questionnaires had been completed by 171 practitioners. The response rate per question varied between 26 and 98%. Of the 260 patients, slightly more were female (58%) than male (42%). The age of the patients varied between 18 and 91 years, and the age distribution was statistically similar among female and male patients. The sample involved nearly three times as many maxillary teeth (74%) than mandibular teeth (26%). In the maxilla, there were fewer molars (15%) than premolars (38%) and incisors and canines (47%), and in the mandible, there were fewer incisors and canines (8%). Table 1 shows the distribution of the types of posts and cores: Slightly fewer teeth had been restored with cast post and cores than with prefabricated posts and direct cores. Of the 114 cast post and cores, 57% had been cast in a gold alloy, 25% in a silver–palladium alloy, and 18% were unaccounted for. Of the 130 prefabricated posts used in combination with a direct core, 51% were titanium posts, 20% were stainless steel posts, 8% were carbon fiber-reinforced resin composite posts, and 21% posts were either of an alternative or unknown material.
Table 1Distribution of type of post and cores among failed restorationsType of post and coreNumberCast post and core114Prefabricated post with direct core130Prefabricated post with cast core5One-piece post crown7Total256
Of the 260 failed post-retained restorations, 54% comprised tapered posts, 22% comprised parallel-sided posts, 20% comprised posts of a combined tapered-parallel design, and 4% comprised posts with a design that was not accounted for. The majority of the cast posts (76%) and of the stainless steel prefabricated posts (73%) were tapered. The titanium posts were equally distributed between the three designs (38, 31, and 31%), and the carbon fiber-reinforced resin composite posts were either parallel sided (50%) or of a combined design (50%).
Of the 176 failed post-retained restorations that were specified as regard to the maximum diameter of the post, 68% comprised posts with a diameter equal to or less than one third of the diameter of the root, and 32% comprised posts with a diameter of more than one third. Of the 237 failed post-retained restorations that were specified as regard to length of post, 76% comprised posts with a length of more than 50% of the total length of the root. Table 2 shows the distribution of the posts as regard to type, material, and length of posts: For all posts, except those of stainless steel, the majority of the posts were of a length that was equal to or more than half the length of the root. For the stainless steel posts, 50% were shorter than half the length of the root.
Table 2Distribution of posts of failed restorations as regards post type, post material, and post length in percent of root lengthTypeMaterialLengthTotal15–49%50–74%75–90%CastGold9391260Silver–palladium318829PrefabricatedTitanium14301761Stainless steel1311125Carbon fiber0729Total3910540184
Of the 130 teeth with a failed restoration that had been treated with a prefabricated post and a direct core, 29% had a core made in amalgam, 59% a core made in resin composite, 3% a core made in glass ionomer cement, and 9% a core made in a material that was not accounted for. Amalgam had been used more often than resin composite for stainless steel posts (70 vs 30%), whereas resin composite had been used more often than amalgam for titanium posts (84 vs 16%) and, in all cases, for carbon fiber-reinforced resin composite posts.
Of the 260 failed post-retained restorations, 40% had been luted with zinc phosphate cement, 17% with glass ionomer cement, 10% with resin cement, and 33% posts with an agent that was not accounted for. The majority of the cast posts (68%) and of the stainless steel prefabricated posts (93%) had been luted with zinc phosphate cement. Most titanium posts had been luted with zinc phosphate cement (48%) or glass ionomer cement (37%). Of the carbon fiber-reinforced resin composite posts, 90% had been luted with resin cement and 10% with glass ionomer cement.
Of the 260 failed restorations 69% had a ferrule, 29% did not have a ferrule, and 2% were not accounted for. As regards the degree of tooth destruction before luting of the now failed post-retained restoration, only 25% restored teeth out of 260 were accounted for. In 47 cases, the whole clinical crown had been missing at the time of restoration, and in 18 cases, between one half and three fourths of the clinical crown had been missing.
Table 3 shows for how long the restorations had functioned in the mouth at the time of failure. The time of functioning of the 178 restorations that were accounted for varied between 3 months and 38 years. The mean survival time until failure was 11 years, and the median survival time was 8 years. Of the 178 restorations, 61% had functioned 10 years or less at the time of failure. Of the 260 restorations, 51% displayed fracture of the tooth, 30% displayed loosening of the post, 17% displayed fracture of the post, and 2% displayed a type of failure that was not accounted for.
Table 3Distribution of failed post-retained restorations as regards functioning time until failureFunctioning time (years)Number0–1152–5506–104311–204321–3827Total178
Among the post-retained restorations that failed after a relatively short functioning time (0–10 years) as compared to a relatively long functioning time (11–38 years) were: (1) significantly more parallel-sided and combined tapered-parallel posts than tapered posts, (2) significantly more carbon fiber-reinforced resin composite prefabricated posts than titanium and stainless steel prefabricated posts, (3) significantly more cast silver–palladium posts than cast gold posts, (4) significantly more resin composite cores than amalgam cores, and finally (5) significantly more posts luted with resin cement or with glass ionomer cement than with zinc phosphate cement. These results are unexpected, and as the “Discussion” will argue, the results do not truly reflect factors critical to functioning time.
Several factors were significantly related to type of failure. (1) The frequency of tooth fracture was similar for female and male patients (51 vs 50%) as was the frequency of loosened posts (36 vs 25%), but the frequency of fractured posts was higher for male than for female patients (24 vs 13%). (2) The shorter the post, the lower the frequency of post fracture (Table 4). (3) Tapered posts more often than parallel-sided and combined tapered-parallel posts displayed fracture of tooth (61 vs 39 and 41%) and less often fracture of post (9 vs 30 and 31%). (4) Stainless steel prefabricated posts more often than titanium prefabricated posts displayed fracture of the tooth (81 vs 36%). (5) Teeth restored with amalgam cores displayed mainly tooth fractures (70%), teeth restored with cast gold cores displayed mainly tooth fractures (51%) or loosening of post (35%), and teeth restored with resin composite cores displayed all three types of failure with almost similar frequency (35, 25, and 40%). (6) More teeth in which the post had been luted with zinc phosphate cement displayed tooth fracture than teeth for which glass ionomer cement or resin cement had been used (54 vs 34 and 36%). (7) Compared with the frequencies of post fracture and loosening of the post, the frequency of tooth fracture increased with increasing functioning time until failure, and correspondingly, restorations that failed early on showed an equal frequency of the three types of failure (Table 5).
Table 4Distribution of failed post-retained restorations as regards length of post and type of failureLength (mm)FailureTotalFracture of toothFracture of postLoosening of post2–520211336–1077204714411–201212731Total1093465208Table 5Distribution of failed post-retained restorations as regards functioning time of restoration and type of failureFunctioning time (year)FailureTotalFracture of toothFracture of postLoosening of post0–1546152–5141511406–10188174311–20272144321–38183526Total823253167
Discussion
Five percent of the private practitioners that had been invited to participate in the survey did so. Possible explanations for this low response rate include the open invitation design, the relative extensive questionnaire, and the fact that the questionnaire could not be completed once and for all but had to be procured whenever a patient presented with a failed post-retained restoration. Considering that prosthodontics is not a registered specialty in Denmark, there is no reason to believe that the treatments provided by the participating practitioners were of another type or quality than those provided by private practitioners in general. The fact that the questionnaires were filled out by individual practitioners in their everyday environment and not by calibrated investigators in an optimal research facility explains that the response rate to each question was not 100% and also implies some uncertainty as to the validity of the answers, especially those that included an element of estimation, e.g., of post diameter. The abovementioned factors call for some caution when interpreting the results of the survey.
As regards age and gender, the patient sample is not only representative of the private practice patient population in Denmark as such but also similar to patient samples in previously published clinical studies of post-retained restorations [9, 27, 46]. These clinical studies also supply explanations for the present finding that more maxillary teeth, especially incisors and canines, than mandibular teeth presented with failed post-retained restorations: Not only are more maxillary teeth than mandibular teeth treated with posts [9, 11, 26, 46], but post-retained restorations in maxillary teeth have also been reported to fail relatively more frequently than similar restorations in mandibular teeth [46]. In particular, post-retained restorations in anterior maxillary teeth have been found to show high failure rates, presumably because of higher functional horizontal forces acting on the anterior teeth than on posterior teeth [13, 26, 27, 46].
The types of posts, cores, and cements used for the failed restorations, not only reflect that preferences vary among practitioners but also that the age of the restorations vary widely, as did the materials and treatments available at the time of restoration. Given the design of this survey, i.e., we have only considered restorations that have failed, we have no knowledge of the entire population of post-retained restorations. Thus, the survey does not allow distinction between an over-representation of a given material or treatment caused by outspread use and over-representation caused by poor longevity.
The length of time that the post-retained restorations had functioned before failure varied from 3 months to 38 years with a mean survival time until fracture of 11 years. In agreement with the findings of previous surveys of failed post-retained crowns [21, 50], there was a tendency to a relatively high initial failure rate. However, the proportion of restorations that failed during the first 10 years was lower in the present survey (61%) than in the survey by Lewis and Smith (94%) [21]. In the survey of Turner [50], only 17% had survived more than 5 years. The longer lifespan found in the present survey may reflect that the samples of the previous surveys included posts that were generally shorter and more often smooth and/or tapered than the posts of the present survey. These features are likely to have increased the prevalence of loosened posts at an early stage.
A number of specific types of posts or materials were found to have significantly high prevalence in the group of post-retained restorations that failed after a relatively short functioning time: carbon fiber-reinforced resin composite posts, posts cast in silver–palladium, parallel-sided and combined tapered-parallel posts, resin composite cores, and resin cement and glass ionomer cement. Some of these posts and materials are probably found in this group of restorations that failed after a short functioning time, not because they in fact are associated with short functioning time but because they have been on the market for a relatively short time compared with the alternatives and thus are over-represented in the total group of restorations with a short functioning time.
The most commonly reported type of failure was tooth fracture followed by loosening of the post and, finally, fracture of the post. This relative distribution is at odds with that of previous, retrospective studies, which found loosening of the post to be the most common type of failure [3, 21, 46, 50]. Failures in the form of loss of retention are most often restorable, whereas tooth fractures are most often nonrestorable and lead to extraction of the tooth. It is possible that it was easier for the practitioner to remember to submit a questionnaire when a more drastic tooth fracture occurred than when a post had lost retention and had to be recemented. Such a situation would tend to underestimate the proportion of loosened posts. An alternative explanation is that the distribution between types of failure has indeed changed to reflect a decrease in the use of smooth and tapered posts.
Several factors were found to significantly influence the distribution between types of failures. One factor was gender of the patient: Whereas there were, relatively speaking, equally many male and female patients in the group of post-retained restorations that displayed tooth fracture or loosening of post, there were more male patients in the group of restorations that displayed post fracture. It may be that the greater bite forces exerted by men [16] not only increase the failure rate of post-retained restorations as such [46] but also predispose for fracture of the post. Another factor was post length: Failed shorter posts were associated with a lower prevalence of post fracture than were longer posts. This finding is not readily explained on the basis of stress analyses but may reflect that there was an over-representation of stainless steel posts and/or of tapered posts in the group of short posts: Stainless steel has a higher elastic modulus and is a stronger material than gold alloys and titanium alloys [52] and may thus be expected to withstand higher stresses. Tapered posts were associated with a higher prevalence of tooth fracture than were parallel-sided and combined tapered-parallel posts and with a correspondingly lower prevalence of post fracture. This significant tendency to fail because of tooth fracture confirms results of previous studies [42, 46]. Likewise, failed stainless steel posts were associated with a higher prevalence of tooth fracture than were titanium posts. This could be an indirect result of most stainless steel posts being short, having high elastic modulus, and having a tapered shape in contrast to most titanium posts being parallel sided. Teeth that failed and had been restored with amalgam cores displayed significantly more tooth fractures than teeth restored with cores of resin composite and cast gold post and cores. Because of intermediate mechanical properties, e.g. elastic modulus, of amalgam as compared to resin composite and gold alloy, mechanical properties cannot easily explain the high prevalence of tooth fracture. Instead, it is likely to be caused not by the amalgam core itself but by other factors that characterize the posts with which amalgam was used. Amalgam was the preferred core material for stainless steel posts, whereas resin composite was the preferred core material for titanium posts. As just mentioned, stainless steel posts were more often than other posts tapered, a factor that has also been shown to predispose for tooth fracture. The fact that zinc phosphate cement also seemed to predispose for tooth fracture may similarly be explained, not by inferiority of the material itself but by the posts with which it was used: A relatively higher percentage of posts cast in silver–palladium and of prefabricated stainless steel posts had been luted with zinc phosphate cement than with any other cement, and these two types of posts were associated with a high prevalence of tooth fracture. The final factor that was found to be significantly associated with type of failure was the length of time that the post-retained restorations had functioned until failure: The prevalence of tooth fracture increased with increasing functioning time until failure. This finding is in contrast to that of previous surveys of failed post-retained crowns in which no correlation between type of failure and length of functioning time was found [21, 50]. The discrepancy can be explained by the fact that the present survey included teeth of a much wider range of functioning time until failure and higher mean age at failure.
This survey did not confirm the generally accepted, paramount influence on fracture prognosis of extent of remaining tooth structure and of a ferrule [9, 12, 30, 42, 44, 45, 47, 54]. With regard to extent of remaining tooth structure, the reason is that an exceptionally low response rate did not allow statistical analysis of this parameter on a possible influence on functioning time and type of failure. The fact that the survey also did not find a significant influence of the existence of a ferrule calls for the following comments. First, the fact that most restorations were reported to have a ferrule reduced the probability that the statistical analysis would find a significant effect. Second, it is very positive to note that clinicians are aware of the importance of including a ferrule in the preparation. Third, the fact that restorations failed despite that the crowns had a ferrule points to the relevance of optimizing other elements in the restoration of an endodontically treated tooth.
Conclusions
The following are the conclusions derived from the study:
Fracture of the tooth was the most common type of failure among the failed post-retained restorations followed by loosening of the post and fracture of the post.Compared with parallel-sided posts, tapered posts more often displayed fracture of the tooth and less often fracture of the post.Post-retained restorations that had functioned for a long time before failure showed an increased risk of tooth fracture compared to loosening of the post and fracture of the post.Fracture of the post was more common among male patients than among female patients.

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fixed prosthodontics


Alterations in aortic elasticity in noncompaction cardiomyopathy

Background Noncompaction cardiomyopathy (NCCM) is a recently recognized disorder frequently associated with systolic and diastolic heart failures. This study was designed to examine aortic stiffness in NCCM patients and to compare these results to age- and gender-matched controls.
Introduction
Noncompaction cardiomyopathy (NCCM) is a recently recognized rare disorder [1, 2]. It is characterized by prominent myocardial trabecularizations, and deep intertrabecular recesses leading to the spongy appearance of the myocardium [3]. The disease is frequently associated with systolic and diastolic heart failure (HF), ventricular arrhythmias, and systemic embolization [4].
Alterations in arterial function have been demonstrated in patients with HF [5, 6]. With two-dimensional transthoracic echocardiography (TTE), ascending aortic diameter changes during a heart cycle can be measured. When blood pressure data are also available, aortic elasticity can be characterized [7, 8]. This study was designed to examine aortic stiffness in NCCM patients and to compare these results to age- and gender-matched controls.
Methods
Study population
A total of 20 patients with typical echocardiographic features of NCCM were investigated [3, 9]. Their clinical and demographic data are presented in Table 1. Clinical assessment included medical and family history, physical examination, electrocardiography, two-dimensional echocardiography, and, in most cases, contrast echocardiography. Their results were compared to 20 age- and gender-matched controls without apparent cardiovascular disease. Informed consent was obtained from each patient, and the study was approved by the institutional review board and complied with the Declaration of Helsinki.
Table 1.Clinical and demographic data of NCCM patientsNCCM patientsAge (years)38 ± 16Male (%)8 (40)Diabetes (%)1 (5)Index eventArrhythmia (%)6 (30)Heart failure (%)8 (40)Screening (%)6 (30)ElectrocardiogramAtrial fibrillation (%)2 (10)LV hypertrophy (%)3 (15)Left bundle branch block (%)6 (30)NCCM noncompaction cardiomyopathy
Diagnostic criteria for NCCM
Previously proposed echocardiographic diagnostic criteria for NCCM by Jenni et al. [3] were used: (1) absence of coexisting cardiac anomalies, (2) segmental, excessive thickening of the left ventricular (LV) wall with a two-layered structure: a thin, compacted epicardial layer and a much thicker, noncompacted layer with the characteristic appearance of numerous, prominent trabeculations (meshwork) and deep intertrabecular recesses, (3) color Doppler evidence of deeply perfused intertrabecular recesses, and (4) predominant localization of thickening in the LV apical, midlateral, and midinferior walls. Hypertensive heart disease was excluded by clinical and echocardiographic examinations (septal thickness < 13 mm).
Blood pressure measurement
Systolic and diastolic blood pressures (SBP and DBP, respectively) were measured in the supine position with an automatic mercury cuff sphygmomanometer from the left arm after 10 min of rest. The first and the fifth Koratkoff sounds were taken for the SBP and DBP. Blood pressure values were averaged from three consecutive measurements. None of the patients or controls used coffee or tea within 1 h before blood pressure measurements.
Transthoracic echocardiography
All patients underwent a complete two-dimensional TTE and Doppler study using a Philips Sonos 7500 echocardiography equipment (Philips, Best, The Netherlands) in the left lateral decubitus position from multiple windows. The LV wall segments were analyzed according to the 9-segment model as described by Jenni et al. [3]. Systolic and diastolic ascending aortic diameters (SD and DD, respectively) were recorded in M-mode at a level of 3 cm above the aortic valve from a parasternal long-axis view (Fig. 1). Gain, depth, and sector angles were individualized for the best measurement. The SD and DD were measured at the time of maximum anterior motion of the aorta and at the peak of the QRS complex, respectively.
Fig. 1Measurements of systolic (SD) and diastolic (DD) diameters of the ascending aorta are shown on the M-mode tracing obtained at a level 3 cm above the aortic valve
Evaluation of aortic stiffness
Aortic stiffness index (β) was defined as ln(SBP/DBP)/[(SD - DD)/DD], where ln is the natural logarithm.
Statistical analysis
Data are reported as means ± standard deviation. For variables, Student’s t test and analysis of variance (ANOVA) test were used where needed. A value of p < 0.05 was considered statistically significant. SPSS 12.0 software was used for statistical calculations.
Reproducibility of echocardiographic measurements
The reproducibility of the aortic diameter measurements was tested in all NCCM patients at both systole and diastole by two independent, blinded observers. Agreement between the two observers was verified using the Bland–Altman method [10].
Results
Patient population
The presenting symptoms were HF in eight (40%) and arrhythmias in six (30%) patients. The remaining six (30%) asymptomatic patients were NCCM relatives and were diagnosed after family screening (Table 1). Three HF patients were in NYHA-class III HF and the other five were in NYHA-class II. Cardiac medication used in NCCM patients were: angiotensin-converting enzyme inhibitors in 13 (65%), beta-blockers in 11 (55%), oral anticoagulant therapy in 10 (50%), diuretics in 8 (40%), digitalis in 2 (10%), and nitrate in 1 (5%). None of the controls received any cardiac medication.
Transthoracic echocardiography
The number of noncompacted segments in the NCCM patients was 4.6 ± 2.0 in the NCCM group and (as seen in Table 2) these patients had significantly increased LV dimensions and reduced LV ejection fraction. Aortic stiffness index (β) was significantly increased in NCCM patients compared to controls. For controls, NCCM patients with moderate HF, and NCCM patients with severe HF, β values were 3.5 ± 1.1, 7.9 ± 5.5, and 10.4 ± 1.8, respectively (p < 0.001). Aortic stiffness indices of all individual patients and controls are presented in Fig. 2.
Table 2.Transthoracic echocardiographic and blood pressure data in NCCM patients and normal subjectsGroup 1 (normal subjects)Group 2 (NCCM patients)LV end-diastolic diameter (mm)47.4 ± 3.661.0 ± 10.9*LV end-systolic diameter (mm)29.9 ± 2.948.2 ± 12.3*LV ejection fraction (%)67.5 ± 5.936.0 ± 17.6*Systolic aortic diameter (mm)26.7 ± 4.126.6 ± 4.4Diastolic aortic diameter (mm)23.3 ± 3.824.6 ± 4.3Pulsatile change in aortic diameter (mm)3.4 ± 1.12.0 ± 1.2*Systolic blood pressure (mmHg)125.2 ± 12.9120.4 ± 17.4Diastolic blood pressure (mmHg)77.8 ± 8.574.1 ± 9.6Aortic pulse pressure (mmHg)47.4 ± 8.746.3 ± 11.7Aortic stiffness index (β)3.5 ± 1.18.3 ± 5.2*Continuous variables are given as mean ± standard deviationLV left ventricular, NCCM noncompaction cardiomyopathy*p < 0.001Fig. 2Individual β indices of NCCM patients and control subjects. NCCM noncompaction cardiomyopathy
Reproducibility
The mean ± standard deviation difference in values obtained by two observers for the measurements of aortic diameter at systole was −0.7 ± 2.2 mm, with a correlation coefficient between these independent measurements of 0.88 (p < 0.01) (Figs. 3a and 4a). At diastole, the difference between these observations was 0.05 ± 1.95 mm, with a correlation coefficient between observations of 0.9 (p < 0.01). The difference in values that were detected by observers was within twofold of the standard deviation of the mean (Figs. 3b and 4b).
Fig. 3a Interobserver correlation (r = 0.88, p < 0.01) between aortic systolic diameters in NCCM patients. b Interobserver correlation (r = 0.9, p < 0.01) between aortic diastolic diameters in NCCM patients. NCCM noncompaction cardiomyopathyFig. 4a Reproducibility of systolic aortic measurements in NCCM patients. b Reproducibility of diastolic aortic measurements in NCCM patients. NCCM noncompaction cardiomyopathy
Discussion
NCCM is a recently recognized disorder characterized by a loosened, spongy myocardium associated with a high incidence of progressive systolic and diastolic HF. In recent studies, alterations have been demonstrated in arterial function in patients with chronic HF [11–14]. To the best of authors’s knowledge, this is the first time that the aortic distensibility in a series of NCCM patients was examined. In this study, increased aortic stiffness index (β) was found in NCCM patients compared to age- and gender-matched controls.
In prior studies, it has been shown that pulsatile changes in ascending aortic vessel diameter can be indirectly measured during routine TTE. Stefanadis et al. [7] found that noninvasive measurements of aortic distensibility were as accurate as invasive methods. Aortic stiffness index (β) relies on aortic and blood pressure data and is one of the most frequently used parameters to characterize arterial stiffness [7, 8].
Increased aortic stiffness is an independent risk factor, predictor of cardiovascular mortality, and a contributor to LV afterload [15, 16]. Increased aortic stiffness occurs early during the development of pacing-induced congestive HF in animals [17, 18] and has also been described in clinical patients with HF [5, 6], with comparable changes in arterial distensibility in ischemic and idiopathic dilated cardiomyopathy [19]. Rerkpattanapipat et al. [11] demonstrated that the distensibility of the proximal aorta is markedly reduced in older patients with HF due to LV systolic dysfunction beyond the changes from the aging process. They also described a correlation between exercise intolerance and reduced aortic distensibility. Giannattasio et al. [12] described that arterial compliance is impaired in congestive HF, and although more marked in severe congestive HF, the impairment is manifested in mild congestive HF as well [12]. Nakamura et al. [13] described vascular hypertrophic remodeling and endothelial dysfunction-associated alterations in vascular wall elastic properties in limb muscle conduit arteries in patients with congestive HF. Poelzl et al. [14] described an intriguing relationship between phenotype changes and functional impairment in peripheral conduit arteries of HF patients. This process is very similar to what is known for ventricular remodeling in HF.
There are several potential factors affecting aortic function in patients with HF [11]. Neurohormonal changes associated with HF include an increase in sympathetic drive and an activated renin–angiotensin system resulting in increased plasma norepinephrine levels causing vasoconstriction and sodium retention [20–23]. Angiotensin II has a hypertrophic effect on smooth muscle cells of the vascular wall [24]. Early atheromatous changes and endothelial dysfunction can also be associated with HF [25], and can affect vascular elasticity. However, there is also an interaction between aortic stiffening and HF. Aortic stiffening can increase LV load causing LV stiffening with increased wall tension, early impairment in diastolic LV relaxation, and contractility inducing LV hypertrophy and fibrosis [26, 27]. These results suggest that aortic stiffness may contribute to the progression of systolic and diastolic LV dysfunctions.
Study limitations
During this study, brachial cuff pressure measurement instead of a direct assessment of aortic pulse pressure by catheter was used. However, previous studies demonstrated an excellent correlation in aortic distensibility measured by invasive and noninvasive methods [7]. Coronary artery disease is correlated with increased aortic stiffness and was not excluded in most but not in all our patients by coronary angiography. The aortic elastic properties of control patients were somewhat larger than those described in the literature. The reason for this can be the larger body mass index (29.0 ± 5.1 kg/m2) in our controls. However, a relatively higher β in the normal subjects in our study only strengthens the abnormal findings in the NCCM patients.
Conclusions
Increased aortic stiffness can be observed in patients with NCCM with moderate to severe HF. These alterations may be due to HF-induced neurohormonal changes.

noncompaction
aortic stiffness
echocardiography


The effects of laparoscopic cholecystectomy, hysterectomy, and appendectomy on nosocomial infection risks

Background Recent reviews of the literature have concluded that additional, well-defined studies are required to clarify the superiority of laparoscopic or open surgery. This paper presents precise estimates of nosocomial infection risks associated with laparoscopic as compared to open surgery in three procedures: cholecystectomy, appendectomy, and hysterectomy.
With the realization of smaller incisions, better cosmesis, less postoperative pain, same-day surgery, speedier postoperative recovery, and the potential for reduced complications, laparoscopic approaches have all but replaced the traditional laparotomic alternatives for certain commonly performed surgical procedures. However, the widespread adoption of laparoscopic techniques into the overall surgical armamentarium has been slowed by a variety of factors, including the learning curves required to integrate new levels of depth perception and fine dexterity, longer operating times without commensurate economic reward, and the nullification of savings from earlier hospital discharge by the cost of disposable surgical instrumentation. Nevertheless, the laparoscopic approach is now widely accepted as the gold standard for cholecystectomy and the surgical treatment of gastroesophageal reflux [1, 2].
Laparoscopic appendectomy has been controversial since its introduction in the early 1980s, particularly for cases of complicated appendicitis. Although laparoscopic appendectomies now account for almost half of appendectomies in the United States [3], several recent reviews of the literature have concluded that additional, well-defined studies will be required to clarify the superiority of laparoscopic or open approaches [4–6].
Despite the demonstration that abdominal hysterectomy is associated with higher morbidity and worse outcomes when compared to the vaginal or laparoscopic approach, the majority of hysterectomies worldwide are still performed in this fashion. For the remaining cases, laparoscopic hysterectomy is least apt to be performed [7, 8]. Although laparoscopy facilitates vaginal hysterectomy for the larger uterus, allows for the concurrent diagnosis and treatment of benign pelvic conditions such as endometriosis or pelvic adhesions, permits concomitant adnexal surgery, and provides the ability to secure and reaffirm intraperitoneal hemostasis at the end of the procedure [9], a meta-analysis of randomized controlled trials comparing different types of hysterectomy published by the Cochrane Collaboration failed to clearly demonstrate the superiority of laparoscopic hysterectomy over vaginal hysterectomy [10].
Some studies have reported that laparoscopic approaches are associated with lower risks of surgical site infections than their open counterparts [11–13], but the effects of laparoscopic surgery on overall nosocomial (hospital-acquired) infection risks have not been established. Since a significant number of nosocomial infections in surgical patients occur after discharge [14, 15], it is likely that comparisons of laparoscopic and open surgeries have underestimated risks.
Nosocomial infections are a leading cause of death in the United States, affecting two to three million patients annually [16]. Starting in 2009, Centers for Medicare and Medicaid Services (CMS) will stop reimbursing hospitals for certain complications including surgical-site infections, catheter-associated urinary tract infections, and central-line associated bloodstream infections [17]. Therefore, the establishment of nosocomial infection risks in general is important.
The objective of this study is to obtain more precise estimates of nosocomial infection risks associated with laparoscopic and open approaches for cholecystectomy, appendectomy, and hysterectomy. We hypothesize that laparoscopic surgery will reduce the risk of nosocomial infections for each of these surgical modalities. To test these hypotheses we performed a retrospective analysis of more than 11,000 admissions, each with one of the procedures of interest, from 22 hospitals that have implemented a nosocomial infection monitoring system that can detect nosocomial infections up to 30 days post discharge.
Methods
The Nosocomial Infection Marker (NIM)
The Nosocomial Infection Marker (NIM, patent pending, Cardinal Health) monitors and tracks nosocomial infection rates for hospitals and communities. Cardinal Health extracts data from client facilities on an ongoing basis using a secure, Health Insurance Portability and Accoutability Act- (HIPAA) compliant method. Data are cleaned and mapped in real time as they arrive at the Cardinal Health data center by proprietary software systems. Rare exceptions that are not electronically modeled are modeled by technical and clinical experts, processed and loaded. The new models are then used by the systems to process like data in the future.
The Nosocomial Infection Marker is a computer algorithm that identifies the existence of nosocomial infections at the microbiological level. Specifically, the NIM algorithm distinguishes likely pathogens from contaminants, identifies duplicate isolates, and temporally determines hospital versus community-acquired pathogen acquisition [18].
In a multihospital study using comprehensive medical records review and gold-standard infectious disease physician discrepancy resolution, the NIM algorithm identified nosocomial infections with 86% sensitivity and 98.5% specificity hospital-wide, statistically outperforming Centers for Disease Control (CDC) case finding methods [18]. Unlike the NIM, traditional CDC case finding methods are subjectively applied with inconsistent results and are only used for certain types of infections, mostly in ICUs [18, 19]. Like the NIM, the performance characteristics of CDC methods have only been formally evaluated in one study [19].
Data
The Nosocomial Infection Marker (NIM) was used to identify nosocomial infections during hospitalization and post discharge. Data were extracted from the Cardinal Health (CAH) data repository for the period September 1, 2004 through December 31, 2006 from 22 hospitals in 15 states. Hospitals with matching International Statistical Classification of Diseases and Related Health Problems (ICD-9) procedure codes and more than 100 admissions with a primary ICD-9 procedure for cholecystectomy, appendectomy, or hysterectomy were eligible. These hospitals had a median number of beds of 359, with an interquartile range from 191 to 483 beds; one hospital exceeded 1,000 beds and two had fewer than 150. Mean hospital CMI was 1.56 with a standard deviation of 0.22. Admissions with primary Diagnosis Related Group (DRGs) listed in Table 1 comprised more than 95% of eligible admissions. When these admissions were restricted to adults 18 years and older for cholecystectomy and hysterectomy, and patients 2 years and older for appendectomy, 11,662 admissions were available for analysis.
Table 1DRGs included in the analysis% admissions NIM rate% laparoscopySimple presentations (complexity = 0)166Appendectomy W/O complicated Principal Diag W Cc3.162.7167.21167Appendectomy W/O Complicated Principal Diag W/O Cc14.531.1271.66195Cholecystectomy W C.D.E. W Cc0.5214.7516.39196Cholecystectomy W C.D.E. W/O Cc0.210.0025.00197Cholecystectomy Except By Laparoscope W/O C.D.E. W Cc3.649.4311.79198Cholecystectomy Except By Laparoscope W/O C.D.E. W/O Cc2.012.5640.60358Uterine & Adnexa Proc For Non-Malignancy W Cc11.433.3036.53359Uterine & Adnexa Proc For Non-Malignancy W/O Cc25.721.2047.92493Laparoscopic Cholecystectomy W/O C.D.E. W Cc14.864.15100494Laparoscopic Cholecystectomy W/O C.D.E. W/O Cc11.420.68100Complex presentations (complexity = 1)164Appendectomy W Complicated Principal Diag W Cc2.969.2847.25165Appendectomy W Complicated Principal Diag W/O Cc3.393.0454.43354Uterine, Adnexa Proc For Non-Ovarian/Adnexal Malig W Cc2.248.055.36355Uterine, Adnexa Proc For Non-Ovarian/Adnexal Malig W/O Cc2.042.1019.33357Uterine & Adnexa Proc For Ovarian Or Adnexal Malignancy1.8910.005.00Diag = Diagnosis; W/O = Without; C.D.E. = Common Duct Exploration; Proc = Procedure; Malig = Malignancy; W = With; Cc = Complication and comorbidities
Data elements included NIMs, age, gender, insurance type (Medicaid, Medicare, private, other), hospital case mix index (CMI), primary DRG, whether or not the admission was through emergency department (ED), and ICD-9 procedure codes. The primary ICD-9 procedure code was used to identify both procedure (cholecystectomy, appendectomy, or hysterectomy) and type of approach (open or laparoscopic) and the primary DRG was used to differentiate simple from complex presentations in an attempt to account for intrinsic infection risks and biases towards open approaches. DRGs indicating malignancy or other complex presentations were assigned a complexity value of 1, as shown in Table 1. Hospital CMI was used to control for differences between and clustering within hospitals.
Statistical Analysis
Single and multiple logistic regression analyses were performed to quantify the associations between NIM rate and procedure, approach, patient age, gender, insurance type, complexity of presentation, ED admission status, and hospital CMI. The first model pooled all three procedures and included binary variables to adjust for the influence of each procedure on the acquisition of NIMs. Then separate models for cholecystectomy, appendectomy, and hysterectomy were constructed. Finally models were constructed for procedure and approach for wound, urinary tract, bloodstream, and respiratory tract NIMs.
Results
Hysterectomies comprised 43.3% of all procedures, cholecystectomies 32.7%, and appendectomies 24.0%. The percentage of cholecystectomies, appendectomies, and hysterectomies that were laparoscopic was 84.7%, 65.6%, and 39.5%, respectively. Unsurprisingly, fewer than one-quarter of all patients were male. Approximately 19.3% of admissions were covered by Medicare, 7% by Medicaid, 58.8% by private health insurance, and the remaining 14.8% by other types of insurance.
NIM rates were defined as the number of admissions with at least one NIM divided by the total number of admissions. Of the 11,662 admissions, 337 (2.89%) had at least one NIM (Table 2). Overall, NIM rates were higher for open approaches (4.09%) than laparoscopic ones (2.11%). NIM rates were highest for cholecystectomy (3.57%), followed by appendectomy (2.60%), then hysterectomy (2.53%).
Table 2Nosocomial infection rates by approach and procedureAdmissionsAdmissions with ≥1 NIMRate (%)11,6623372.89Approach  Laparoscopic70611492.11  Open46011884.09Procedure  Cholecystectomy38081363.57  Appendectomy2803732.60  Hysterectomy50511282.53Approach by procedure  Laparoscopic    Cholecystectomy3226842.60    Appendectomy1840422.28    Hysterectomy1995231.15  Open    Cholecystectomy582528.93    Appendectomy963313.21    Hysterectomy30561053.44
There were 399 NIMs identified in 337 admissions. Of all NIMs identified, 118 (30%) were from surgical wounds, 122 (31%) were from the urinary tract, 37 (9%) were from the blood, 29 (7%) were from the respiratory tract, and 93 (23%) were from other sources.
At least one post-discharge NIM was identified in 136 admissions, accounting for 40% of all admissions with a NIM. Of the 147 post-discharge NIMs, 39% were from surgical wounds, 31% were from the urinary tract, 7% were from blood, and 22% were from other sources. Of the 136 total admissions with at least one post-discharge NIM, 92 patients had NIM-associated readmissions.
Univariate Analyses
Simple logistic regressions examined associations between NIM rates and surgical approaches (laparoscopic or open), and the following covariates: gender (male, female), age (<18 y, 18–34 y, 35–49 y, 50–64 y, 65–74 y, ≥75 y), surgical procedure (cholecystectomy, appendectomy, hysterectomy), type of insurance (private, Medicare, Medicaid, other), complexity of admission on presentation (0/1), admitted through ED (0/1), and hospital CMI. The results, summarized in Table 3, show significantly higher NIM rates for males, adults aged ≥65 years, patients undergoing cholecystectomy, complex admissions, and admissions covered by Medicare; and significantly lower NIM rates for laparoscopic surgery, females, adults 18–49 years old, patients undergoing hysterectomy, and those covered by private insurance. NIM rates were positively correlated with hospital CMI, but were unaffected by ED admission status.
Table 3Univariate analyses of factors associated with NIMVariable CategoryNIM rate (%)OR95% CIGenderMale3.841.501.19–1.90Female2.59Age<18 years2.410.820.49–1.3918–34 years1.500.460.32–0.6535–49 years2.200.660.52–0.8450–64 years3.411.250.97–1.6165–74 years3.891.411.02–1.95≥75 years7.102.962.25–3.90InsurancePrivate2.110.510.42–0.65Medicare5.242.321.85–2.92Medicaid3.191.120.74–1.68Other2.770.950.70–1.29ApproachLaparoscopic2.110.320.21–0.52Open4.09ProcedureCholecystectomy3.571.411.13–1.76Appendectomy2.600.870.67–1.13Hysterectomy2.530.790.64–0.97CMI2.281.59–3.27ComplexityComplex6.312.742.14–3.50Not complex2.40Emergency department admissionEmergent3.031.060.72–1.55Nonemergent2.88CI: confidence interval; NIM: nosocomial infection marker; OR: odds ratio
Multivariable analyses
Since ED admission status was insignificant in the univariate analysis, it was excluded from the multivariable analyses. Pairwise correlations of all remaining covariates were performed, and all pairs were reasonably uncorrelated. Therefore, all were included in the multivariable analyses.
Results of multiple logistic regression models, which controlled for gender (male, female), age (<18 yr, 18–34 yr, 35–49 yr, 50–64 yr, 65–74 yr, ≥75 yr), type of insurance (private, Medicare, Medicaid, other), complexity of admission on presentation (0/1), and hospital CMI, show that laparoscopic procedures reduced the odds of acquiring a nosocomial infections by half, but that the effect is entirely attributable to reduced infection risks in laparoscopic cholecystectomy and hysterectomy with odds ratios (ORs) of 0.34 (p < 0.01) and 0.48 (p < 0.01), respectively. No change in nosocomial infection risk was found for laparoscopic appendectomy. Estimates for individual procedures are shown in Table 4.
Table 4Multivariable logistic regression analyses of factors associated with NIMVariableOdds ratio for NIMPooled (n = 11,662)Cholecystectomy (n = 3,808)Appendectomy (n = 2,803)Hysterectomy (n = 5,051)Laparoscopy0.48**0.34**0.970.48**Type of procedure    Cholecystectomy1.87**–––    Hysterectomy1.05 –––Age    <18 years0.83–0.90–    18–34 years0.64*0.470.840.72    50–64 years1.222.13*1.000.96    65–74 years1.02*2.21*0.580.51    ≥75 years1.92**4.04**3.310.61Male1.4*1.111.89*–Type of insurance    Medicare1.421.331.122.09*    Medicaid1.453.47**1.110.79    Others1.291.531.221.32    CMI1.63*1.091.312.88*    Complexity2.45**NS3.952.54**** Statistically significant at the 1% level* Statistically significant at the 5% level
Consistent with results of the univariate analyses, multivariable regression showed a significantly higher risk of NIMs for males, patients aged ≥65 years, and complexity of presentation at the time of admission. CMI was found to be  significant in both univariate and multivariable analyses [OR = 1.63 (CI95 1.11–2.40), p < 0.05], and this risk was significantly higher for hysterectomy patients, for whom the odds of acquiring a NIM were as high as 2.88 times that of not acquiring NIM.
Analysis of the same dataset by source of infection (urinary tract, wounds, respiratory tract, bloodstream, and others) revealed that the overall infection rates at each of these sites were all statistically significantly lower for laparoscopic approaches, as summarized in Table 5. The odds of acquiring a site-specific infection were statistically significantly lower for all sites in laparoscopic cholecystectomy and for wound sites in hysterectomy.
Table 5Odds ratios by sourceUrinary tractWound Respiratory tractBloodstreamOthersOverall OR (95% CI)0.61 (0.38–0.96)0.41 (0.27–0.62)0.20 (0.08–0.49)0.31 (0.14–0.65)0.52 (0.33–0.82)By procedure    Cholecystectomy0.48 (0.24–0.97)0.20 (0.11–0.39)0.17 (0.06–0.45)0.23 (0.10–0.55)0.34 (0.18–0.64)    Appendectomy0.83 (NS)1.06 (NS)0.27 (NS)Too few NIMs0.91 (NS)    Hysterectomy0.76 (NS)0.27 (0.09–0.79)Too few NIMs 0.48 (NS)0.62 (NS)OR: odds ratio, CI: confidence interval, NS: not significant
There were no significant differences between laparoscopic versus open appendectomy for all sources of nosocomial infections (urine, blood, wound, respiratory, and other). However, laparoscopic appendectomy is associated with a statistically significantly higher risk of abscess (p < 0.05), a finding consistent with the literature [12].
Forty-one percent of patients with a nosocomial infection had at least one post-discharge nosocomial infection, and 58 of 115 (50%) of surgical wound infections were post discharge. There were 118 readmissions associated with at least one post-discharge NIM, and post-discharge NIMs associated with readmission were significantly lower for laparoscopic approaches (p < 0.001). Excluding appendectomy, the odds ratio for laparoscopic versus open NIM-associated readmission was 0.346 (CI95 0.19–0.63).
Discussion
This study demonstrates that laparoscopic cholecystectomy and hysterectomy reduced the overall odds of acquiring nosocomial infections from all sources by more than 50% and reduced the odds of readmission with nosocomial infection by two-thirds. Laparoscopic appendectomy showed no differences in overall nosocomial infection risks compared to open surgery. The findings for wound infections are consistent with results from randomized trials, which have reported statistically significantly lower surgical site infection rates for laparoscopic approaches [10–12, 20].
This study also demonstrates statistically significant differences in source-based infection risks by procedure and approach. Specifically, wound, bloodstream, respiratory tract, urinary tract, and other nosocomial infections were all statistically significantly less likely to occur in association with laparoscopic cholecystectomy. Risks of wound infections in laparoscopic hysterectomy were also significantly lower than in open procedures. However, no differences in infection risks were found between laparoscopic and open appendectomy.
It was important to stratify admission by complexity of presentation to avoid biases associated with complex presentations, higher intrinsic infection risks, and surgical approaches; for example, primary DRGs indicating malignancy or complex presentations (Table 1) were associated with open surgical approaches in 69% of admissions, whereas simple presentations were associated with open approaches in only 35% of admissions. Complex presentations are also commonly believed to be at higher risk of infections, an association that is also demonstrated in this analysis. Therefore, by controlling for presentation complexity, this analysis accounts for some of the intrinsic risk of infection as well as a bias towards open surgical approaches.
The difference in patient severity between hospitals was accounted for by using CMI, and indeed CMI is significant in the univariate and multivariable models, with CMI contributing to nosocomial infection risks. Interestingly, admission through the emergency department was not significant in determining differences in nosocomial infection risks. One possible explanation is that emergency department use is a crude measure of patient severity because it may also be related to other factors such as time of day of admission and socioeconomic status. However, other variables associated with comorbidity, like age and certain payer types, were significant.
Limitations
While a variety of confounders were controlled for, this study is limited by the absence of certain data; for example, antibiotic use, anesthesia scores, wound class, body mass index, prior hospitalization, and certain comorbidities (i.e., cardiovascular status, diabetes mellitus, and immunodeficiency) were unavailable. These variables could explain additional NIM risk. Although omitted-variable bias is often a concern in multivariable modeling using retrospective databases, the similarity of findings in our univariate and multivariable analyses suggest that these results are robust.
This study documents for the first time that laparoscopic hysterectomy and cholecystectomy are associated with statistically significantly lower overall risks of nosocomial infections (p < 0.01). Laparoscopic hysterectomy and cholecystectomy were also associated with statistically significantly fewer readmissions with nosocomial infections (p < 0.01). Differences in infection risks between laparoscopic and open appendectomy were not found to be statistically significant, suggesting that these differences are likely small or nonexistent. Where differences in risks have been demonstrated, future studies should be performed to quantify their effects on health care costs and length of stay.
Other important directions for future research include controlling for potentially important confounders to test the robustness of our results and extending the analysis to examine the effect of laparoscopic versus open surgery on the risk of nosocomial infections for additional surgical procedures. Another interesting avenue for future research would be to examine the effect of hospital volume on the rate of nosocomial infections.

laparoscopic cholecystectomy hysterectomy appendectomy nosocomial


Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm−2 trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL−1 horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.
Introduction
A demand for smaller enzyme reactors has emerged in recent years, as a consequence of ongoing miniaturization in the biochemical and analytical sciences. These micro-reactors have been used in biocatalysis and biosensing. In the field of proteomics the reactors are a tool in peptide mapping, in which proteins are identified via peptide fragment identification after proteolysis. Currently, in spite of its limitations, most of these analyses are conducted by means of 2D gel electrophoresis followed by digestion of the proteins, liquid chromatographic (LC) separation, and mass spectrometric (MS) identification of the peptides [1–3]. The most time-consuming step in this procedure is digestion of the protein using a protease. In general, every protein to be investigated is individually incubated with the protease at a concentration of approximately 1–2% protein weight for 2 to 18 h at an elevated temperature (typically 37 °C). In addition to the long incubation time needed, a certain level of auto-digestion of the protease can be expected. To reduce sample handling, digestion time, and the risk of sample contamination, methods for the on-line digestion of proteins have been developed that use proteases immobilized on a solid support.
Immobilized enzyme reactors have been developed and used over the years for several industrial and analytical purposes [4–6]. An obvious benefit for immobilizing biocatalysts is the fact that the enzyme can be used in several catalytic cycles and that both catalyst and reaction mixture can easily be separated. Moreover, immobilized enzymes generally show an improved stability even at more extreme reaction conditions. Several procedures have been developed for immobilization of enzymes, e.g. adsorption or encapsulation in a matrix or membrane. Alternatively, and more often used, is the covalent attachment of biocatalysts to carrier materials, which allows the immobilization of a large amount of enzyme for a high activity per surface area. Generally, particulate large-pore carrier materials are used, such as controlled-pore glass [7, 8], silica [9], or polymers like the commercially available poroszyme [10–12]. Current research in the production of immobilized enzymes is focused on the use of monolithic materials, as they enable efficient fragmentation of proteins [13–17]. Although both commercially available and self-prepared reversed-phase capillary monolithic columns have successfully passed reproducibility assessment [18, 19], synthesis of monoliths suitable for small-scale enzyme reactors can still be troublesome. Materials suitable for the fabrication of larger-scale enzyme reactors are commercially available from BIA Separations (Ljubljana, Slovenia).
Although it is possible to apply an immobilized enzyme reactor (IMER) positioned after the separation column [20], most papers dealing with on-line digestion of protein samples position the IMER upstream of the separation column. In these cases the sample is first digested and the resulting peptide fragments are separated and identified by LC–MS. This approach is often employed in multi-dimensional LC methods [13, 21, 22], and has also found application in peptide mapping using capillary electrophoresis [23, 24]. Alternatively, as recently shown by Zhao et al. [25] and Krenkova et al. [26], who covalently coupled trypsin to the wall of fused-silica nanoelectrospray emitters, a protein sample can be analyzed by direct infusion into a mass spectrometer.
This paper describes the development of trypsin-modified open-tubular micro-reactors. The chemistry was controlled and optimized using surface plasmon resonance (SPR), a technique allowing sensitive and real-time monitoring of surface reactions such as protein binding [27]. The surface modification resulting in the highest enzyme immobilization yield, was used to covalently immobilize the trypsin on the inside wall of a fused-silica capillary. The constructed trypsin micro-reactor, which is compatible with micro- and nano-LC, was further characterized. The influence of reaction time, pH, temperature, and reactor stability were investigated with the model substrate insulin B-chain. The reactor was also applied to digestion of the proteins cytochrome C and myoglobin. The produced peptides were analyzed by liquid chromatography–mass spectrometry.
Experimental
Materials
The SPR equipment used was from IBIS Technologies (currently available from Eco Chemie, Utrecht, The Netherlands) equipped with a 200-μL polycarbonate cuvet. The gold-sensor disks, purchased from SSENS (Hengelo, The Netherlands), were positioned on the IBIS-prism using index-matching oil from R.P. Cargille Laboratories (Cedar Grove, USA). PEEK nuts, unions, tubing, and loops were from Upchurch (Santa Monica, USA). Manual injections during the preparation of the reactors were performed using a Rheodyne 7010 injector (Inacom, Veenendaal, The Netherlands) equipped with a 1-mL PEEK loop. Model 10ADvp HPLC pumps from Shimadzu (Kyoto, Japan) were used for reactor preparation and activity determinations. The water used for washing and to prepare buffers was produced by a Sartorius Arium 611 ultrapure water system (Nieuwegein, The Netherlands; conductivity >18.2 MΩ cm). The model ABS759A UV absorbance detector was equipped with a capillary flow-cell (75-μm i.d.) and was obtained from Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands).
On-line digestion experiments with micro-HPLC separations were conducted using LC-Packings instruments and columns (Amsterdam, The Netherlands). The equipment consisted of an injector (Famos), nanovalve column switcher (Switchos), nanopump (Ultimate), and a photodiode-array detector (PDA) equipped with a micro flow cell (45 nL). The reversed-phase pre-columns were 5 × 0.3 mm with 5-μm 100 Å C18 particles. The 150 × 0.3 mm reversed-phase micro-column contained 3-μm 100 Å C18 PepMap particles. The mass spectrometer was an Agilent LC/MSD XCT ion trap (Amstelveen, Netherlands).
Acetic acid, boric acid, calcium chloride (CaCl2), ethanol, ethanolamine (EA), hydrochloric acid (HCl), hydrogen peroxide, sodium dihydrogen phosphate, sodium chloride, sodium hydroxide (NaOH), and sulfuric acid were purchased from Merck (Darmstadt, Germany). HPLC grade acetonitrile and ethanol were from Biosolve (Valkenswaard, The Netherlands). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris-hydroxymethylaminoethane (TRIS), urea, and acetone were purchased from Acros Organics (Geel, Belgium). Benzamidin, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), benzoyl-arginine ethyl ester (BAEE), iodoacetamide, N-hydroxysuccinimide (NHS), and carboxyl-modified dextran (CMD) were purchased from Fluka (Buchs, Switzerland). Horse-heart cytochrome C, insulin B (oxidized), polyoxyethylenesorbitan monolaurate (Tween 20), and porcine pancreas trypsin were purchased from Sigma (St Louis, USA). The specific activity of trypsin was determined according to the method of Schwert and Takenaka [28] using BAEE as substrate and turned out to be 14700 U mg−1. Aminopropyltriethoxysilane (APTES), carbonyl diimidazole (CDI), glycidoxypropyltrimethoxysilane (GOPS), and mercaptoethanol (ME) were from Aldrich Chemical Company (Milwaukee, USA). Amino-modified dextran (AMD) was from Unavera ChemLab (Mittenwald, Germany). Fused-silica capillaries were purchased from Bester (Mijdrecht, The Netherlands).
Piranha solution was prepared by mixing 1 part 30% hydrogen peroxide and 6 parts concentrated sulfuric acid (caution, aggressive solution). TRIS digestion buffer consisted of 50 mmol L−1 TRIS and 1 mmol L−1 CaCl2 adjusted to pH 8.2 by use of 1 mol L−1 HCl solution.
Methods
Preparation of dextran-coated SPR sensor disks (Fig. 1)
Fig. 1
Surface modifications used to immobilize the enzyme in fused-silica capillaries. a, GOPS-modified; b, GOPS/AMD-modified; c, APTES/CMD/AMD-modified surface. In the last case the CDI activation and enzyme (H2N–enzyme) coupling are as in b
The SPR sensor disks were extensively cleaned in freshly prepared piranha solution. After 1 h the disks were thoroughly rinsed with water, dried in a stream of nitrogen gas, and immediately incubated for 6 h in a 10 mmol L−1 solution of ME in ethanol in order to produce a self-assembled monolayer (SAM) containing hydroxyl functionalities. After SAM formation, the disks were washed with ethanol and water, and dried with nitrogen gas. The resulting ME disks were incubated for 1 h with a 10% GOPS solution in 98% ethanol after which the disks were washed with ethanol, dried with nitrogen gas and stored overnight at 50 °C.
Dextran-modified sensors were prepared by incubating GOPS-modified sensor disks for 20 h at room temperature with a 10% AMD solution in a 50 mmol L−1 borate buffer, pH 9.5. After removing the solution the disks were washed with water, ethanol, and water, dried in a gentle flow of nitrogen gas, and stored at room temperature in a closed box until use.
Alternatively, ME disks were incubated for 1 h with a 10% APTES solution in acetone. After washing, drying, and storing overnight in the oven, similar to the GOPS disks, the surfaces were incubated for 1 h with a solution containing 5% CMD in water containing 200 mmol L−1 EDC and 50 mmol L−1 NHS. After removing the solution, the disks were washed with water and ethanol, and incubated for 15 min in a solution containing 100 mmol L−1 CDI in acetone. These activated surfaces were washed with ethanol, blown to dryness with nitrogen, and incubated overnight with a solution of 10% AMD in water. After removing the solution the disks were washed with water, ethanol, and water, dried in a gentle flow of nitrogen gas, and stored at room temperature in a closed box until use.
SPR experiments
The GOPS-modified SPR sensor disks were incubated overnight with 200 μL of a solution containing 2.5 mg mL−1 trypsin and 50 μg mL−1 benzamidin in 50 mmol L−1 borate buffer, pH 9.5 (Fig. 1a). Both types of dextran-modified sensor disk were activated for 60 min with 200 μL 100 mmol L−1 CDI in dry acetone. After washing the surface with water to remove the last traces of acetone, the sensors were incubated overnight with 200 μL 2.5 mg mL−1 trypsin and 50 μg mL−1 benzamidin in 50 mmol L−1 borate buffer, pH 8.5 (Fig. 1b and c, respectively). The remaining esters were inactivated by incubating the disks for 10 min with 1 mol L−1 EA in 50 mmol L−1 borate buffer, pH 8.5. The successive steps in the immobilization were monitored with SPR and the amount of covalently coupled enzyme was calculated from the recorded angle shift. During the experiments the SPR system was thermostatted at 25.0 °C.
Preparation of dextran-coated fused-silica capillaries
In order to generate a proper surface for silanization, fused-silica capillaries were cleaned for 30 min with 2 mol L−1 NaOH solution at a flow-rate of 5 μL min−1. The capillary was then washed for 30 min with 0.1 mol L−1 HCl, for 5 min with water, and finally for 5 min with ethanol. In order to prepare dextran-coated capillaries, the capillaries were flushed for 60 min with a 10% GOPS solution in ethanol. After this step, the capillaries were closed with silicon plugs and dried overnight at 50 °C. After silanization, the capillaries were flushed with methanol at a flow-rate of 10 μL min−1 after which the capillaries were chemically modified in flow, using injections with a Rheodyne 7010 manual injector equipped with a 1-mL PEEK loop, in a way similar to that described above in the SPR section. Washing steps were conducted at flow rates of 10 μL min−1, overnight incubations at a flow rate of 1 μL min−1. All chemistries are outlined in Fig. 1.
Enzyme activity determination
Insulin B chain was used as a substrate to determine the activity of trypsin. Injections of 1 μL of a concentration of 20 μmol L−1 in digestion buffer were introduced into the reactor that was kept at the indicated temperature using a water bath. To determine the enzyme activity in solution, trypsin was incubated in these solutions also (final trypsin concentration 0.4 μg mL−1). At time intervals from 0.5 to 30 min, 100-μL samples were taken and the activity was stopped by the addition of 5 μL 20% TFA in water. Buffers that were used to determine the effect of pH on activity were MES (pH 5.5 to 6.5), MOPS (pH 6.5 to 7.8), TRIS (pH 7.5 to 9), and CHES (pH 8.6 to 10.1). The buffers were adjusted to pH by use of 1 mol L−1 NaOH and were prepared at a 50 mmol L−1 concentration, also containing 5 mmol L−1 CaCl2. To determine the effect of temperature, the temperature during incubation was varied between 10 °C and 60 °C. Both the on-line and off-line samples were analyzed using micro-HPLC with PDA detection, as outlined below. The insulin B conversion of both immobilized trypsin and the enzyme in solution was calculated from the peak areas of substrate and products.
On-line protein digestion in micro-HPLC
For the on-line peptide and protein digestion experiments the trypsin-modified capillaries were positioned between the LC injector and a 10-port valve, as shown in Fig. 2, and were immersed in a thermostatted water bath set at 37 °C unless mentioned otherwise. Protein samples were prepared in digestion buffer and were transported through the capillary towards a 10-port valve using a 5% acetonitrile solution containing 0.05% TFA. The peptide fragments formed during digestion were concentrated on an RP trapping column (TC) and salts and other buffer components present in the sample were removed. By switching the valve the trapping column is in series with the RP micro column and an acetonitrile gradient started. The gradient was composed of two solutions: (A) 5% acetonitrile in water containing 0.05% TFA and (B) 80% acetonitrile in water containing 0.04% TFA. In 30 min the gradient changed linearly from 0 to 50% B, followed by 10 min at 90% B and 20 min at 0% B. The eluent was monitored with the PDA detector in the range from 200 to 595 nm.
Fig. 2Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used
ESI-MS was conducted in the positive-ion mode with the capillary voltage set at 3500 V. The flow rate and temperature of the nitrogen drying gas were 5 L min−1 and 325 °C, respectively. The sequence of the peptide fragments was determined by using the mass spectrometer in auto-MS–MS mode fragmenting the two peptides that were most abundantly present when the signal reached threshold. The MS–MS result was analyzed by a Mascot database search (http://www.matrixscience.com).
Results and discussion
Surface chemistry and enzyme immobilization
As shown before [29], modification of an SPR sensor surface with a dextran hydrogel leads to less non-specific adsorption of proteins compared with unmodified surfaces. The presence of such a layer also enhances the immobilization capacity of biomolecules compared with monolayer-based coatings. Due to the flexible nature of the dextran chains, the accessibility also is often improved compared with molecules immobilized on a flat surface. As the amount of protein present in a capillary after immobilization cannot be determined easily, SPR sensors were used as a model to investigate the effect of the different surface modifications on the amount of trypsin that could be attached covalently. Hydroxyl functionalities necessary to enable silanization are introduced using mercaptoethanol (ME), but all other surface modifications are carried out in exactly the same way, both on the SPR sensor surfaces and in the fused-silica capillaries.
Trypsin immobilization after silanization with GOPS by reaction of the trypsin primary amines and the glycidyl function of GOPS results in an SPR angle shift of 350 ± 20 m° (n = 3), which equals an amount of trypsin of 2.9 ± 0.2 ng mm–2 covalently attached to the surface, which is close to monolayer coverage of trypsin. When the GOPS-silanized surface is modified with AMD, resulting in a dextran hydrogel, the amount of trypsin that can be immobilized after CDI activation increases to 9.5 ± 0.3 ng mm−2 (n = 3). To further increase the amount of trypsin, an intermediate dextran layer was added. Therefore, fused-silica capillaries were APTES silanized and modified with CMD and AMD, subsequently. The amount of trypsin that could covalently be attached to these layers was determined with SPR and proved to be 11.1 ± 0.5 ng mm−2 (n = 2). As this amount is significantly more than obtained with the GOPS/AMD surface, all further experiments have been conducted with fused-silica capillaries the surface of which is modified with dextran in this way.
Assuming that the investigated surface modification allows a similar amount of trypsin to be immobilized per surface area in fused-silica capillaries, the quantity of enzyme immobilized on the dextran-modified capillaries is more than five times larger per surface area compared with other open-tubular, trypsin micro-reactors described in the literature [23, 30–32]. These reactors, first described by Amankwa and Kuhr [30], are based on immobilized avidin and, at saturation, 6.5 pmol biotinylated trypsin was immobilized in a 50-cm long capillary of 50-μm i.d.. This amount equals 2 ng trypsin mm−2 capillary surface, which is less than monolayer coverage.
Characterization of the enzyme reactor
Fused-silica capillaries were modified with dextran as described above and the activity and characteristics of the trypsin micro-reactor were assessed with the oligopeptide insulin B chain, a 30 amino acids long insulin fragment. When insulin B is exposed to the enzyme present in the reactor, hydrolysis at the arginine present at position 22 and, to a lesser extent, the lysine at position 29 is observed. Using the aforementioned substrate insulin B, the pH optimum was determined for the enzyme in solution and the immobilized enzyme. As can be observed in Fig. 3a, the optimum pH value for both free and immobilized trypsin is pH 8.5. Therefore, all further experiments are conducted at this pH value.
Fig. 3Effect of (a) pH and (b) temperature on the relative activities of trypsin in solution (circles) and trypsin immobilized on an APTES/CMD/AMD-modified fused-silica surface (triangles). In b the maximum insulin B conversion rate determined under experimental conditions is 4.41 ± 0.02 pmol min−1 μg−1 for the enzyme in solution and 12.32 ± 0.35 pmol min−1 μg−1 for a 3-μL, 50-μm i.d. microreactor operated at a flow rate of 1 μL min−1
Also the effect of temperature on the activity of both the immobilized trypsin and the enzyme in solution was determined in the range 10 °C to 60 °C. As can be observed in Fig. 3b, the activity of trypsin increases with temperature to reach a maximum around 40 °C and decreases at higher temperatures. The immobilized enzyme shows a higher activity compared with the enzyme in solution, which may be due to an often observed higher stability of immobilized enzymes, also under more extreme conditions. From the data the activation energy can be determined. For the immobilized enzyme a value of 12.2 ± 0.3 kJ mol−1 can be calculated and for the enzyme in solution a value of 17.2 ± 0.5 kJ mol−1. This means that for the immobilized enzyme the temperature has less effect on the activity than for the enzyme in solution. As the highest activity is observed at 37 °C, all further experiments are conducted at that temperature.
For the enzyme in solution a specific activity of 4.41 ± 0.02 pmol min−1 μg−1 is determined at a concentration of 20 pmol μL−1 insulin B and pH 8.5 and 37 °C. Under similar conditions, in a microreactor of 3 μL with a 50 μm i.d. and operated at a flow rate of 1 μL min−1, an amount of 12.32 ± 0.35 pmol insulin B is converted. In such a reactor a total of 2.67 μg trypsin might be present taking the earlier SPR observations into account, which means that the immobilized enzyme is both accessible and still active after immobilization. The specific activity after immobilization is higher than observed in recent papers for the protease pepsin immobilized on beaded chitosan [33, 34] and trypsin photoimmobilized in a fused-silica capillary [34].
The stability during operation of the open-tubular reactor was tested at 37 °C and pH 8.5. Activity tests using the model substrate show that the activity of a reactor that is continuously in operation, is constant for at least two weeks. The enzyme trypsin dissolved in digestion buffer and incubated for 24 h at 37 °C loses 60% of its activity, and after three days no activity is measured.
On-line digestion of proteins
The on-line digestion of horse cytochrome C is accomplished as described in the Methods section. Samples are submitted to on-line digestion in a 22-cm long trypsin-modified APTES/CMD/AMD-coated capillary with an i.d. of 75 μm (total volume 1 μL) or with an i.d. of 50 μm and a length of 51 cm (1 μL) or 140 cm (2.75 μL). When the protein is not reduced, the heme-moiety will remain covalently attached to the peptides containing the protein sulfhydryls. By monitoring the heme-containing peptides at 395 nm, the progress of the digestion and the amount of undigested protein can be determined. The results are summarized in Table 1.
Table 1Summary of the on-line digestion experiments for cytochrome C. The experiments were conducted at pH 8.5 and 37°C (n = 3)capillary ID (μm)reactor volume (μL)amount injecteddigestion time (s)% undigested protein (SD)(μg mL−1)(pmol)751248206065.9 (4.8)751124106035.8 (3.475112.4106023.6 (1.3)501248206014.0 (1.9)50112410602.3 (2.0)502.75248201650502.75124101650502.7512.4101650The experiments were conducted at pH 8.5 and 37 °C (n = 3)
As expected when a limited amount of enzyme activity is present in a reactor (22 cm  ×  75 μm), for increasing concentrations cytochrome C a larger amount of protein is undigested. Nevertheless, many tryptic peptides are still generated. As can be expected, an increase in exposure time of the substrate with the immobilized enzyme will result in an improved digestion yield. A longer contact time is achieved by increasing the reactor volume by using a longer enzyme-modified capillary. Additionally, the enzyme-to-substrate ratio is increased, which is accomplished by changing the surface-to-volume ratio by using a capillary with a smaller internal diameter. As can be observed in Table 1, decreasing the i.d. of the capillary leads to improved digestion for a reactor of equal volume due to a higher surface-to-volume ratio and hence a higher amount of enzyme. The use of a 140 cm ×50 μm capillary allows the complete digestion of up to 20 pmol (248 μg mL−1) of cytochrome C in less than 5 min including the sample concentration and removal of salts by the trapping column.
Chromatograms for the on-line digestions obtained with capillaries with an i.d. of 50 μm are shown in Fig. 4. Figure 4a shows a blank run in a capillary containing no enzyme and Fig. 4b and c present on-line digestions for a micro-reactor of 2.75 μL operated at 5 μL min−1 and 1 μL min−1, respectively. As discussed above, incomplete digestion of the injected cytochrome C will lead to the presence of multiple peptide fragments containing the heme group, as is visible in Fig. 4b. The digestion is complete when the sample exposure time is 165 s (flow rate 1 μL min−1) as both intermediate products and the undigested protein (retention time 44 min in Fig. 4a), which are visible at a wavelength of 395 nm as outlined above, are no longer observed. An injection of off-line-digested cytochrome C showed a similar chromatogram as is shown in Fig. 4c.
Fig. 4Chromatograms obtained from injection of 10 pmol horse cytochrome C in capillary digestion systems monitored at 214 nm and 395 nm. The experiments were conducted with an APTES-CMD-AMD derivatised fused-silica capillary of (a) 510 × 0.050 mm, not containing trypsin, operated at 1 μL min−1 (blank); (b) 1400 × 0.050 mm, trypsin-modified, operated at 5 μL min−1 (average sample residence time 33 s); (c) as (b) but operated at 1 μL min−1 (average sample residence time 165 s). For clarity the beginning of the chromatogram displaying the 214 nm signal is offset as indicated
The on-line digestion of horse cytochrome C is also monitored with mass spectrometry. The effect of flow rate and hence incubation time on the digestion of the protein and the number of peptides identified with a Mascot database search is determined and summarized in Table 2. In this table the undigested amounts of protein which have been determined using the PDA detector are also shown. At a flow rate of 1 μL min−1 protein digestion is complete and many peptides are matched, resulting in high sequence coverage and Mascot score. With higher flow, and hence decreasing incubation time, the amount of protein that remains undigested increases and consequently fewer peptides are produced and observed. However, even at relatively high flow rates an adequate amount of peptides is still formed and the protein can be identified on the basis of the fragments present. Nevertheless, the ion intensity is low and some peptides are not retrieved as they are below the threshold for auto-MS–MS. Table 3 summarizes the peptides observed and matched using the MS–MS data and a Mascot.database search of proteins digested at a flow rate of 1 μL min−1. A base-peak chromatogram (BPC) of the digestion of cytochrome C under these conditions is shown in Fig. 5a. The MS–MS fragmentation of one of the peptides is presented in Fig. 5b.
Table 2General overview of the on-line digestion experiments for cytochrome C (n = 3)flow rate (μL min-1)digestion time (s)sequence coverage (%)total no peptides matchedMascot scoreundigested protein (%)1017661134760.3533662150131.8283662364918.5116587278330Table 3Peptide fragments observed by MS in the on-line digestion of cytochrome C and myoglobin The experiments were conducted at pH 8.5 and 37 °C at a flow rate of 1 μL min−1 using a 2.75-μL trypsin reactorPeptide sequenceTheoretical m/zExperimental m/zPositionMissed cleavageCytochrome C (sequence coverage 87%)1....IFVQKCAQCHTVEK1632.8816.9 (2+)9–2212....HKTGPNLHGLFGR1432.8717.1 (2+)26–3813....TGPNLHGLFGR1167.6584.6 (2+)28–3804....TGPNLHGLFGRK1295.7648.6 (2+)28–3915....KTGQAPGFTYTDANK1597.8533.4 (3+)39–5316....TGQAPGFTYTDANK1469.7735.6 (2+)40–5307....TGQAPGFTYTDANKNK1711.8571.4 (3+)40–5518....GITWKEETLMEYLENPKK2208.1736.8 (3+)56–7329....EETLMEYLENPK1494.7748.0 (2+)61–72010  EETLMEYLENPKK1622.8812.1 (2+)61–73111...YIPGTK678.4678.1 (1+)74–79012...MIFAGIKK906.5454.0 (2+)80–87113...KTEREDLIAYLKK1477.8739.6 (2+)88–99214...TEREDLIAYLKK1349.7675.5 (2+)89–99115...EDLIAYLK963.5482.6 (2+)92–99016...EDLIAYLKK1091.6546.6 (2+)92–1001Myoglobin (sequence coverage 88%)1....GLSDGEWQQVLNVWGK1814.9908.7 (2+)1–1602....VEADIAGHGQEVLIR1605.8803.6 (2+)17–3103....VEADIAGHGQEVLIR1605.8536.1 (3+)17–3104....LFTGHPETLEK1270.7636.0 (2+)32–4205....HLKTEAEMK1085.6543.5 (2+)48–5616....HGTVVLTALGGILK1377.8689.7 (2+)64–7707....HGTVVLTALGGILKK1505.9502.7 (3+)64–7818....KKGHHEAELKPLAQSHATK2109.1703.7 (3+)78–9629....KGHHEAELKPLAQSHATK1981.0661.1 (3+)79–96110  GHHEAELKPLAQSHATK1853.0618.8 (3+)80–96011...YLEFISDAIIHVLHSK1884.0628.8 (3+)103–118012...HPGNFGADAQGAMTK1500.7751.5 (2+)119–133013...ALELFRNDIAAK1359.8680.6 (2+)134–145114...YKELGFQG940.5471.0 (2+)146–1531Fig. 5On-line digestion of 10 pmol cytochrome C at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond to the matched peptides in Table 3, and (b) MS–MS fragmentation of the peptide TGPNLHGLFGR with m/z 584.9 showing several of the matched fragment ions
In further experiments horse myoglobin was digested on-line. This protein is generally regarded as difficult to digest [15, 35]. When 1 μL of a 10 μmol L−1 solution in buffer is injected at a flow rate of 1 μL min−1 (165 s exposure time), the injected protein is completely digested, as was observed with UV detection (data not shown). Using mass spectrometric analysis, 13 different peptides are observed and matched using the MS–MS data and a Mascot database search, resulting in a sequence coverage of 88%. The matched peptides are summarized in Table 3 and a BPC of the on-line digestion of myoglobin is shown in Fig. 6a, with an MS–MS spectrum of one of the tryptic peptides. Both the degree of digestion and the sequence coverage are adequate compared with other systems that often use a high percentage of modifier to enhance digestion, as the absence of denaturing agents during digestion leads to little or no digestion of myoglobin [35]. Therefore these reactors are generally used for direct infusion into MS or off-line protein digestion as the presence of high concentrations of methanol or acetonitrile in the digestion buffer will seriously impede on-line protein digestion in combination with RP-LC.
Fig. 6Results from on-line digestion of 10 pmol myoglobin at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond with the matched peptides in Table 3, and (b) the MS–MS spectrum of the peptide HGTVVLTALGGILK with m/z 689.7
Amankwa and Kuhr [30] reported that proteins and peptides adsorb to the capillary surface of their trypsin reactor. Reactor fouling, and as a result sample carry-over, is also observed when trypsin-modified particulate polymers or monoliths are used [36]. Adsorption of proteins and peptides to these relatively hydrophobic columns is prevented by the addition of up to 20% methanol to the digestion buffer. For dextran-coated surfaces, it is known that the non-specific adsorption of protein is minimal and depends on the character of the oligosaccharide layer [29, 37, 38]. During protein-digestion experiments on SPR sensor surfaces modified identically with the capillary reactors no adsorption during incubation is observed (data not shown). Similarly, during on-line digestion using the dextran coated capillaries, sample adsorption and carry-over are not observed, as repeated sample injections result in identical chromatograms and blank buffer injections do not show peptide fragments. On basis of the latter result and the long lifetime of the reactor, trypsin auto-digestion is probably absent.
The developed IMER has been applied for the digestion of horse cytochrome C and myoglobin in buffer without prior pretreatment followed by separation and MS–MS sequencing. The time needed to obtain complete protein digestion and sample trapping is 5 min with an average sample residence time of 165 s. The total analysis time, digestion and LC separation, is 65 min. The developed micro-reactor is competitive when compared with other systems used for on-line protein digestion described in literature [13, 14, 39–41]. In the capillary IMERs described by Amankwa and Kuhr [30] and Bossi et al. [34] the complete digestion of native proteins was only obtained when the protein resided in the trypsin-coated capillary for 15–25 min. The peptide separation was carried out with CE in these latter cases.
Many reactors use a protein denaturation step to make the protein more susceptible to proteolytic action. The digestion efficiency can be enhanced by partial denaturation by addition of 35–45% acetonitrile to the buffer [11, 15, 35, 36] or by sonication [42, 43]. However, the latter has not yet been used in an automated set-up and the presence of high amounts of modifier will make reversed-phase chromatography in a subsequent step for the separation of the fragments produced during protein digestion very difficult. The described reactors were efficient in terms of time needed for digestion and the digestion result, but were, as a rule, used for direct infusion into the MS or used for off-line digestion.
Alternatively, protein denaturation is accomplished using chemicals such as SDS, guanidine HCl or urea. A recent example is the miniaturized on-line proteolysis–capillary LC system as described by Samskog et al. [12], who employed a 10-μL column packed with poroszyme. The sample contained guanidine.HCl for protein denaturation, and was diluted with buffer, also containing methanol, prior to injection. The total time needed for digestion, trapping, and removal of the high concentration of salt was 15 min. The system needed periodic regeneration to counteract the effects of residual salts in the analyte.
Conclusions
To study the immobilization of the protein trypsin in a fused-silica capillary, a number of surface modifications was tested. In order to investigate the amount of protein that can be covalently attached to these surfaces, SPR sensor disks are modified with these coatings to mimic the capillary surface. The SPR measurements show that the best results were obtained using a dextran coating with an intermediate layer. The resulting open-tubular trypsin reactors having a pH optimum of pH 8.5 display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously.
The capillary reactors show flow-dependent catalysis. For a capillary IMER, the conversion of the insulin B chain increases with decreasing flow and hence a longer residence time. The same is observed for the digestion of horse cytochrome C. The complete digestion of 20 pmol μL−1 horse cytochrome C is observed without the need of protein denaturation, reduction, or alkylation when the average residence time of the protein sample in a 140 cm  ×  50 μm capillary IMER is 165 s. For the proteins used in this study trypsin-reactors were described that were capable of faster digestion using denaturing agents like acetonitrile to enhance the digestion process. However, the presence of such agents would seriously hamper direct analysis of the peptides formed using RPLC and MS. Identification of the proteins cytochrome C and myoglobin is possible by the tryptic peptides that are produced on-line, separated by micro-RPLC, and analyzed using mass spectrometry with auto MS–MS.
The open-tubular reactor can be produced easily, reproducibly, and inexpensively, and can be used for other applications such as enzyme-inhibitor studies. Future research will focus on the development of miniaturized multi-dimensional analysis systems employing on-line digestion using these IMERs. The capillary enzyme reactors show no backpressure and seem promising for coupling to other analytical techniques such as capillary electrophoresis and surface plasmon resonance.

trypsin reactor
on-line digestion
surface plasmon resonance
dextran hydrogel
liquid chromatography