Attenuation-Corrected vs. Nonattenuation-Corrected 2-Deoxy-2-[F-18]fluoro-d-glucose-Positron Emission Tomography in Oncology, A Systematic Review

Purpose To perform a systematic review and meta-analysis to determine the diagnostic accuracy of attenuation-corrected (AC) vs. nonattenuation-corrected (NAC) 2-deoxy-2-[F-18]fluoro-d-glucose-positron emission tomography (FDG-PET) in oncological patients.
Introduction
The attenuation of photons originating from the subject before they are detected by the camera is a generic limitation of nuclear medicine imaging. This attenuation can lead to image distortion and impairs adequate quantification. Attenuation correction has been commonly employed in 2-deoxy-2-[F-18]fluoro-d-glucose (FDG) imaging in an attempt to correct for these effects. With positron emission tomography (PET) scanners, this is accomplished by transmission scanning using a radionuclide source, such as germanium-68 or cesium-137, and with PET/computed tomography (CT) using CT. With respect to visual interpretation of the images, the added value of attenuation correction has been controversial. Whereas attenuation correction provides a more realistic image of FDG distribution, its application significantly increases acquisition times on standard full-ring (FR) PET scanners. In addition, the performance of attenuation correction can introduce noise and even artifact. Paradoxically, even if the nuclear medicine community sees attenuation correction, or the lack of it, as a potential effect-modifier of test accuracy, its impact is rarely accounted for in systematic reviews on the diagnostic accuracy of PET. As a result, the impact of attenuation correction on lesion detectability and interpretation of PET for oncological purposes is not well established. With PET/CT scanning, it is customary to evaluate either modality (primarily to account for artifacts), but one needs to know how to deal with discrepancies.
The objective of this systematic review and meta-analysis was to determine the diagnostic accuracy of nonattenuation-corrected (NAC) and attenuation-corrected (AC) FDG-PET in oncological patients. We studied the effects of attenuation correction for both FR-PET and dual-head coincidence PET (DH-PET), and as a function of different body locations (head/neck, chest, abdomen/pelvis).
Materials and Methods
Literature Search
A computer-aided literature search was performed in both Medline and Embase databases without time range or language restrictions, applying controlled vocabulary (MeSH and EMTREE keywords, respectively) as well as free text words. The search date was February 10, 2006. The search strategy (Appendix) included terms for PET with FDG, modified from Mijnhout et al. [1] as well as search terms identifying both radionuclide and X-ray transmission, emission, attenuation correction, and oncological studies in humans. In addition, the reference lists of the eligible articles were reviewed to ensure that relevant articles had not been missed.
Study Selection
From the list of retrieved articles, articles were initially evaluated for eligibility on the basis of title and abstract by two independent reviewers (UJ, PR). If there was uncertainty as to whether an article was eligible for inclusion, the entire article was reviewed. Inclusion criteria were (1) clinical studies evaluating FDG imaging with and without attenuation correction in oncology patients; (2) study population of at least ten patients; (3) sufficient detail to reconstruct a 2 × 2 contingency table expressing FDG imaging results by disease status, or sufficient detail to reconstruct relative lesion detection measurement of AC vs. NAC imaging; and (4) studies utilizing FR-PET and/or DH-PET. We excluded abstracts, editorials, and reviews, although the latter two categories were used for cross-referencing.
Methodological Quality Assessment
The methodological quality of each article was independently assessed by each reviewer in terms of internal and external validity (Table 1), based on the Cochrane Methods Group in Screening and Diagnostic Tests, modified for our area of interest [2]. Internal validity items focus on whether a valid reference test was used and whether this reference test was uniformly and independently applied and interpreted as well the type of study design. The external validity items evaluate the applicability of the results in terms of the type of patient population and spectrum, demographics, the inclusion/exclusion criteria, the knowledge of previous test/clinical information that could influence interpretation, and the index test characteristics. Items were scored as positive, negative, or unclear.
Table 1Methodological assessment of individual diagnostic studies: criteriaTestCriteriaA. Internal study validity Al. Valid reference testHistology, AC FR or DH coincidence PET A2. Blind measurement of reference test(s) without knowledge of index testAssessment of reference test independent of index test(s) results A3. Avoidance of verification biasChoice of patients assessed by reference test independent of index test result A4. Index test(s) interpreted independently of all clinical informationMentioned in publication A5. Prospective studyMentioned in publicationB. External study validity B1. Spectrum of diseasesLocalization of disease described (selected or general) B2. Demographic informationAge and sex given B3. Inclusion criteria describedMentioned in publication B4. Exclusion criteria describedMentioned in publication B5. Avoidance of selection biasConsecutive series of patients B6. Standardized execution of index test(s)Described technical aspects of index test(s)C. Reproducibility describedMentioned in publication
Data Extraction and Quantitative Analysis
In addition to methodological quality assessment, data related to the type of camera, the FDG dose, the time interval between injection and imaging, the transmission and emission acquisition protocols, the reconstruction protocol, and the interpretation protocol were independently extracted from each study by each reviewer. For studies where it was possible, a contingency 2 × 2 table was constructed. Disagreements were solved by consensus.
For studies using an independent gold standard (histopathology), we determined the sensitivity and specificity of the index tests using the number of true positive, false positive, true negative, and false negative results from the 2 × 2 contingency table. Furthermore, we calculated the “relative lesion detection,” defined as the percentage of lesions scoring equally positive or negative with NAC vs. AC images. We performed a subgroup analysis for different locations of lesions and analyzed sensitivity, specificity, or relative lesion detection of NAC vs. AC for lesion location in the head and neck region, the chest, and the abdominopelvic region. In cases of discrepancy of relative lesion detection between NAC and AC, we extracted data to analyze whether this was related to lesion size and/or intensity.
The statistical diagnostic heterogeneity of the sensitivity and specificity per index test across studies was tested by the chi-square test. In case of statistical heterogeneity of DH- or FR-FDG-PET imaging, a random effect model for pooling was used, whereas in case of statistical homogeneity, a fixed-effect model was used. Sensitivity, specificity, and relative lesion detection were pooled independently, all pooled estimates are presented with 95% confidence intervals (95% CI). The logit transformed sensitivity, specificity, relative lesion detection, and corresponding 95% CI of the index tests were compared using z-test statistics. A p value of less than 0.05 was considered significant.
All statistical analyses were performed with the SPSS 11.0.01 program for Windows (version 11.0.1., SPSS, Chicago, IL, USA).
Results
The search strategy yielded 2,202 references, 1,477 in Medline and 725 in Embase on February 10, 2006. Of the Embase references, 370 were also included in Medline, leaving a total of 1,832 unduplicated references. On the basis of title and abstract alone, 1,806 references were excluded. After review of the full text of the remaining 26 articles, an additional 11 studies [3–12] proved to be ineligible because they did not perform a direct comparison of the yield of NAC vs. AC images in oncological patients. One study [13] was excluded because it was published in abstract form only. Another study [14] was excluded because it was published in Japanese and was not readily translatable. Finally, the study of Hustinx et al. [15], who evaluated the effect of attenuation correction in abdominal tumors for a sodium iodide crystal (NaI) PET scanner, was excluded because no 2 × 2 contingency tables could be constructed. Eventually, we included 12 studies for review [16–27].
A summary of the methodological quality assessment results can be found in Table 2. Methodological quality was scored as negative when quality items were unclear or absent in the original article. In a minority (4/12 = 33%) of studies, histology served as the reference test. However, nine of the 12 studies provided a direct comparison of AC and NAC PET. In three studies, blind measurement of reference test was performed without knowledge of index test. All but one study avoided verification bias. In four studies, the index test(s) was evaluated independently of all clinical information. All studies provided information about the spectrum of diseases being evaluated and standardized the execution of the index test(s). Almost all studies (11/12 = 92%) described the demographics of the study population and inclusion criteria. However, only one study mentioned specific exclusion criteria. Six studies were prospective. Only two of the 12 studies specifically mentioned including consecutive patients, and only three studies specifically described the reproducibility of their results.
Table 2Quality assessment of included studiesStudyYearA1A2A3A4A5B1B2B3B4B5B6CBleckmann1999++++−+−+−−+−Chan2001+−+−−+++−−+−Delbeke2001+−+−++++−−+−Even-Sapir2004+−+−++++−++−Kotzerke1999+−+−−+++−+++Lonneux1999+−+−+++−−+++Nakamoto2002++++++++−−++Reinhardt2005+−+−−++++−+−Schauwecker2003+−−−−+++−−++Weber1999+−+−++++−−+−Zimny1999++++−+++−−+−Zimny2003+−++++++−−+−
Meta-Analysis
Three FR-PET studies were eligible for pooling of sensitivity on a patient basis [22, 24, 25]. The pooled sensitivities for AC and NAC FR-PET were 64% (95% CI 52–74%) and 62% (95% CI 51–73%), respectively (n = 182 patients). Two FR-PET studies provided data that allowed pooled analysis of specificity [22, 24]. Weber et al. [25] could not be included as there were no patients without disease. The pooled specificities for AC and NAC FR-PET were 97% (95% CI 92–99%) and 99% (95% CI 95–100%), respectively (n = 155 patients). For DH-PET, only one study provided data on sensitivity and specificity [27].
Relative lesion detection for NAC vs. AC PET was pooled for 11 studies, which are demonstrated in Fig. 1. Lesion detection of NAC FR-PET vs. AC FR-PET was 98% (95% CI: 96–99%) for n = 1,012 lesions (pooling of n = 7 studies); 79% of which were classified FDG positive at AC FR-PET. Lesion detection of NAC DH-PET vs. AC DH-PET was 88% (95% CI: 81–94%) for n = 288 lesions (pooling of n = 4 studies); 74% of which were classified as FDG-positive at AC DH-PET.
Fig. 1Pooled lesion detection of NAC vs. AC images for FR-PET and DH-PET.
In addition, we evaluated the relative lesion detection depending on body location (head/neck, chest, abdomen/pelvis) in the four FR-PET [20, 21, 23, 26] and in the three DH-PET studies that provided sufficiently detailed information [17, 18, 26]. The relative sensitivity and specificity based on body location could not be calculated due to an insufficient number of studies. For FR-PET, we found similar relative lesion detection for the three body locations: 95% for head/neck (95% CI 84–98%, n = 61 lesions), 97% for the chest (95% CI 94–99%, n = 396 lesions), and 97% for the abdomen/pelvis (95% CI 93–0.99%, n = 205 lesions). For DH-PET, relative detection rates for NAC were not significantly different for the various body sites: 78% in the abdomen/pelvis (95% CI 65–88%; 53 lesions), 84% in the chest (95% CI 74–91%; 136 lesions), and 90% in the head/neck area (95% CI 73–97%; 38 lesions). However, in chest (p = 0.000089) and abdomen/pelvis (p = 0.0037), the relative detection rates with NAC (vs. AC) for DH-PET were significantly lower than those obtained with FR-PET.
A comprehensive analysis of the potential association of relative detection and lesion size and/or intensity, for lesions with discrepant AC and NAC results, was not possible due to a lack of detailed information. We summarized the results in Table 3: findings of Bleckmann et al. and Reinhardt et al. [16, 23] suggest that AC and NAC discrepancies may relate to (intrapulmonary) lesion size with more discrepancies occurring with smaller lesions at the subcentimeter level (an average of 3% of lesions were correctly detected with NAC and not with AC). The single discrepant lesion in the study of Weber et al. concerned a <1-cm lesion in the mediastinum [25]. However, the discrepant lesions in the studies of Nakamoto et al., Schauwecker et al., and Delbeke et al. included both small- and moderate-sized lesions (in relation to lesions included each study) [18, 22, 24]. In the study of Schauwecker et al., the discrepant lesions demonstrated SUVmax values ranging from 1.8 to 2.6, whereas, in the study of Delbeke et al., the two discrepant lesions demonstrated only mildly enhanced uptake on AC images and equivocal uptake on NAC images.
Table 3Evaluation of discrepant lesions between AC and NAC images with respect to lesion size and intensityStudyCamera typeNumber of discrepant lesionsSize rangeIntensity (semiquantitative or qualitative)Bleckmann et al.FR-PET5<1 cmNot givenNakamoto et al.aFR-PET11.8 cmNot givenReinhardt et al.FR-PET60.5–1.1 cm79/174 lesions demonstrated discrepancy in qualitative lesion intensity: 72/174 lesions demonstrating higher intensity (i.e., better visibility) on NAC imagesSchauwecker et al.aFR-PET40.8–3.9 cm31.8–2.6 (SUVmax)Weber et al.FR-PET10.8 cmNot givenDelbeke et al.DH-PET21.0–3.0 cm“Mild uptake” at AC, “equivocal uptake” at NACaHistology used as gold standard and detailed information given only for true positive lesions
Discussion
The cumulated evidence summarized in this systematic review of oncological FDG imaging studies suggests that the accuracy of attenuation and nonattenuation corrected FR-PET are similar. However, with DH coincidence imaging NAC images detect 12% less lesions than AC images, without prominent differences between body areas.
Although in the nuclear medicine field attenuation correction is generally seen as an important issue, it is surprising to find that several large systematic reviews did not thoroughly consider this as a potential effect-modifier. Gould et al. performed systematic reviews on FDG PET in pulmonary lesions [28] and mediastinal lymph node staging in non-small cell lung cancer [29]: in the former review, the item was not mentioned, and in the latter, attenuation correction was an item of study quality, but no analysis of potential impact was performed.
The choice of the reference test is obviously relevant in studies on test accuracy. In oncology, histopathology is the typical endpoint. Of the 12 eligible studies, four used histology as an independent gold standard. Meta-analysis of sensitivity and specificity was only possible for FR-PET, and we found no significant difference for either measure. We chose to use the AC detection rate as an alternative reference test, which defines the relative lesion detection of NAC vs. AC images. This choice theoretically biases in favor of AC: Bleckmann et al. and Reinhardt et al. [16, 23] reported an average of 3% more true positive lesions with NAC FR-PET. However, we expect that the resulting error is small because, in the comparison with histopathology, false positive rates were quite low for either modality. Despite this theoretical negative bias towards NAC, similar lesion detection rates were observed with both AC and NAC for FR-PET. Hence, attenuation correction may not contribute to the detection of malignancy using FR-PET. Conversely, with DH-PET, AC images demonstrated a significantly higher detection rate as opposed to NAC images, which is surprising given that AC images are usually significantly noisier than NAC images. We postulate that this may be secondary to differences in reconstruction/filtering algorithms.
In addition, there are limitations associated with performing a meta-analysis and data pooling, such as the homogeneity of the data and the quality of the published studies. Homogeneous data have higher statistical strength than heterogeneous data. The data in our study were heterogeneous so that we used a random effect model for pooling. In addition, the statistical strength of the meta-analysis is limited by the quality of the published studies included in it. As mentioned earlier and summarized in Table 2, the studies had several quality limitations. Finally, meta-analyses are limited by publication bias, which biases towards the publication of favorable results or popular subjects.
We were surprised by the limited number of good comparative studies evaluating the value of attenuation correction. It appears that attenuation correction has been accepted as the standard of practice without sound scientific evidence to support it.
The introduction of PET/CT machines has made the time constraints associated with transmission scanning less of an issue. However, PET/CT is not a panacea; X-ray transmission scanning has its own problems and numerous PET/CT publications have demonstrated artifact that can be introduced with X-ray transmission scanning [5, 30–45]. Furthermore, in the study of Reinhardt et al. [23], a significantly improved visibility was demonstrated for 41% of lung metastases with NAC images as opposed to CT-AC images. This higher visibility for NAC images was even more pronounced for lesions smaller than 1 cm. These findings underline that even as PET/CT use becomes more widespread, evaluation of both NAC and AC images should remain an integral part of image interpretation, and not just to recognize image artifacts. At the same time, NAC vs. AC discrepancies at PET/CT offer an obvious opportunity for further investigation.
Conclusions
In this meta-analysis, we found no significant difference in sensitivity, specificity, or relative lesion detectability between AC and NAC FR FDG PET. However, attenuation correction improved lesion detection for DH coincidence imaging.

positron emission tomography
attenuation correction
deoxyglucose (mesh)
tomography, x-ray computed (mesh)
tomography, emission-computed (mesh)
neoplasms (mesh)
human (mesh)
systematic review (mesh)


Pneumomediastinum: a rare complication of anorexia nervosa in children and adolescents. A case study and review of the literature

Spontaneous pneumomediastinum is uncommon in paediatric practice. We describe two cases of spontaneous pneumomediastinum in a child and an adolescent with anorexia nervosa. Thorough investigation failed to reveal any underlying cause for secondary pneumomediastinum. Pneumomediastinum in anorexia nervosa can be caused by not only elevated intrathoracic pressures, but also by the poor quality of the alveolar walls due to malnutrition. The incidence of spontaneous pneumomediastinum in anorexia nervosa is probably higher than that recorded, since it resolves spontaneously and, therefore, it can remain undetected. We conclude that it is our considered opinion that malnutrition associated with anorexia nervosa predisposes for spontaneous pneumomediastinum due to weakness of the alveolar wall and the loss of connective tissue.
Introduction
Pneumomediastinum is rarely associated with anorexia nervosa (AN). Approximately 20 cases of pneumomediastinum in anorexia patients are described in the literature. Vomiting, a common symptom in AN, is a known cause of pneumomediastinum [13]. But of the cases reported in the literature, only a few (three) were preceded by vomiting [3, 4, 12]. So, there has to be another cause placing patients with AN at risk for spontaneous pneumomediastinum. We report one case with spontaneous pneumomediastinum as the presenting symptom of AN. In the other case, spontaneous pneumomediastinum is a complication of AN. A review of the literature is given and we discuss the possible pathophysiology of spontaneous pneumomediastinum in patients with AN.
Case report
Case 1
A 13-year-old girl presented with increasing complaints of an unusual crackling sensation and sound in her neck during three days. At that time, she complained of neck pain and headache.
When these complaints started, she had a sore throat with painful swallowing.
Further medical history mentioned a Cooper test, a test of physical fitness, at school two weeks earlier. There had been no trauma or injury and no coughing or vomiting.
Physical examination showed a respiratory rate of 16 per minute, cardiac rate of 51 per minute, blood pressure of 87/55 mmHg and temperature of 36.5°C. Her height was 150 cm (SD −2), weight 34 kg (SD −1.5) and body mass index (BMI) 15 kg/m2 (SD −2.5). There was a slight bilateral bulging of the skin of her neck from the mandible to both clavicles, with, on palpation, a crackling sensation as a diagnostic sign of subcutaneous emphysema. Apart from being extremely slim, the skin was normal, with no signs of injury. Cardiac evaluation showed normal heart sounds without cardiac murmur. There was no dyspnoea. On auscultation, there was normal bilateral vesicular breathing. Blood tests revealed no abnormalities. Chest X-ray and computed tomography (CT) scan revealed mediastinal emphysema with extension to the neck region (Fig. 1). There was no pneumothorax and no extension to the retroperitoneum. An oesophageal gastric passage X-ray was normal, without signs of perforation. Laryngopharyngoscopy did not reveal mucosal lesions or signs of submucosal swelling. Electrocardiography was normal, except bradycardia. After admission to the hospital, she showed loss of appetite and further history revealed an anorectic episode, for which, she had been referred by her general practitioner to a psychologist. At the age of 13 years, she weighed 46 kg, diminishing to 37 kg 4 months later. At the time of admission to the hospital, her weight was 34 kg at the age of 13 years and 6 months. There was absolutely no history of vomiting.
Fig. 1Coronal computed tomography (CT) reconstruction with mediastinal emphysema and subcutaneous emphysema in both axillas and lower neck regions in patient 1 with anorexia nervosa (AN)
Within a week, the subcutaneous emphysema disappeared. She was referred to an eating disorders clinic to manage her eating disorder. She never showed any signs of purging during treatment and recovered from her AN.
Case 2
A 17-year-old girl with known AN and vomiting presented with extreme malnutrition requiring refeeding. She complained of unusual crackling sensation in her neck during eight days, starting after a choking incident while drinking, followed by coughing. The same day, she felt pressure on her chest. During the following days, her neck became swollen and her voice became hoarse. The swelling had already subsided considerably at the time of presentation.
Physical examination showed an extreme cachectic girl of 17 years with a respiratory rate of 16 per minute, cardiac rate of 64 per minute, blood pressure of 85/55 mmHg and temperature of 36.0°C. Her height was 170 cm (SD=0), weight 34.4 kg (SD<<−2) and BMI 12 kg/m2 (SD<<−2.5).
There were signs of subcutaneous emphysema in the neck and in both arms. She had dry skin without signs of injury. Cardiac auscultation was normal without cardiac murmur. There was no dyspnoea. Pulmonary auscultation was also normal. Blood tests revealed no abnormalities. Chest X-ray and CT scan revealed subcutaneous emphysema in the neck and mediastinal emphysema with extension to the intra-abdominal, retroperitoneal and epidural regions (Fig. 2). There was no pneumothorax.
Fig. 2Chest CT showing epidural pneumatosis (arrow) in patient 2 with pneumomediastinum and AN
Laryngopharyngoscopy did not reveal mucosal lesions. The laryngeal mucosa was slightly swollen.
After refeeding, she was referred to an eating disorders clinic to manage her eating disorder.
Within two weeks, the subcutaneous emphysema further resolved spontaneously.
Discussion
Pneumomediastinum, the presence of free air contained within the mediastinum, usually results from spontaneous alveolar wall rupture and, far less commonly, from disruption of the upper airways or gastrointestinal tract [8].
Spontaneous pneumomediastinum can be distinguished from pneumomediastinum secondary to traumatic events, such as chest trauma or thoracic surgery, endobronchial or oesophageal procedures, mechanical ventilation or other invasive procedures [13].
A large and diverse group of factors has been implicated in the development of spontaneous pneumomediastinum. Various respiratory manoeuvres that have in common the development of high intrathoracic pressures may lead to pneumomediastinum. These include Valsalva manoeuvres, coughing, vigorous crying and vomiting. The mechanism of development of spontaneous pneumomediastinum was originally described by Macklin and Macklin as increased intra-alveolar pressure causing the rupture of alveoli with the escape of air into the mediastinum. The mediastinum is subsequently decompressed by further passage of the air into the retroperitoneal and subcutaneous spaces (the Macklin effect) [1, 9]. Whereas primary pneumomediastinum is a relatively benign and self-limited disorder, secondary pneumomediastinum due to oesophageal perforation or Boerhaave’s syndrome is a potentially life-threatening disorder because of the inevitable development of mediastinitis. Primary pneumomediastinum rarely requires treatment as the alveolair rupture heals and the leaked air resolves spontaneously. By contrast, secondary pneumomediastinum due to oesophageal perforation often requires surgical intervention [8].
Pneumomediastinum typically manifests as sudden chest pain or dyspnoea and, less commonly, with dysphagia and hoarseness [8]. Our first patient, however, presented with a sore throat, painful swallowing, complaints of pain in the neck and headache. The second patient presented with initial chest pain and hoarseness. The detection of subcutaneous air in the neck and anterior chest wall and Hamman’s sign, a crunching sound synchronous with the heartbeat, are important physical findings. Radiographically, pneumomediastinum manifests on the posteroanterior projection as a thin radiolucent line parallel to the outline of the heart, as the mediastinal pleura is displaced laterally, and on the lateral projection as the accumulation of air retrosternally [8].
When chest pain and dyspnoea occur, panic attacks must be differentiated from pneumomediastinum. Panic disorders are often seen in patients with AN [6]. As a consequence, we believe that pneumomediastinum should be ruled out in anorexia patients presenting with signs of a first panic attack.
Spontaneous pneumomediastinum is rarely associated with AN. Only 21 publications related to this association are reported by Pubmed.
Purging behaviour, such as self-induced vomiting, is common among AN patients. Vomiting is a known cause of pneumomediastinum, placing anorexia patients, who frequently vomit, at risk for pneumomediastinum. However, in only a few of the cases described in the literature was vomiting was the preceding event of the pneumomediastinum. In most reports, like in our patients, pneumomediastinum in anorectic patients is not preceded by vomiting [1, 2, 4, 5, 7, 8].
In our patients, the diagnostic procedure did not reveal an oesophageal or upper airway perforation. The pathophysiologic mechanism of air entrance into the mediastinum can be explained by the rupture of an alveolar wall, which is mostly the cause of an air leak into the mediastinum [8]. Based on the literature and our observations, patients with AN are at risk for spontaneous pneumomediastinum, even if they are not vomiting.
We speculate that the state of malnutrition contributes to the risk of spontaneous pneumomediastinum.
Animal studies reveal that calorie restriction causes a loss of alveoli and a fall in gas-exchange tissue [10, 11] and thinner alveolar walls [10]. To survive periods of not eating, organisms sacrifice comparatively non-essential structures for gluconeogenesis to provide glucose for the brain and amino acids to maintain muscle. Because the total organismal and lung oxygen consumption fall during calorie restriction, some lung tissue is expendable [10].
Also in human studies, atrophic changes, such as large alveoli and thin alveolar walls, due to starvation were found in Jewish people in the Warsaw Ghetto [14].
With thinner alveolar walls and the loss of alveoli, malnourished individuals are at risk of alveolar wall rupture.
Since known factors of increased intra-alveolar pressure were absent in our first case and since clinical examination and radiographic study did not reveal an esophageal or upper airway perforation, we must assume that subclinical alveolar leaks with subsequent air dissection, pneumomediastinum and diffuse soft tissue emphysema occurred because of weakness of the alveolar wall and thinning of the connective tissue caused by severe malnutrition. Therefore, even with a minimal increase of intra-alveolar pressure, such as that which may occur during usual daily activities, such as a choking incident, can become the cause of air leaks, as in our second case.

pneumomediastinum
anorexia nervosa
adolescence
childhood


Confirmation of Sentinel Lymph Node Identity by Analysis of Fine-Needle Biopsy Samples Using Inductively Coupled Plasma–Mass Spectrometry

Background The sentinel lymph node (SLN) biopsy technique is a reliable means of determining the tumor-harboring status of regional lymph nodes in melanoma patients. When technetium 99 m-labeled antimony trisulfide colloid (99 mTc-Sb2S3) particles are used to perform preoperative lymphoscintigraphy for SLN identification, they are retained in the SLN but are absent or present in only tiny amounts in non-SLNs. The present study investigated the potential for a novel means of assessing the accuracy of surgical identification of SLNs. This involved the use of inductively coupled plasma–mass spectrometry (ICP-MS) to analyze antimony concentrations in fine-needle biopsy (FNB) samples from surgically procured lymph nodes.
The sentinel lymph node (SLN) biopsy technique, introduced in the early 1990s, has made it possible to establish the tumor-harboring status of the regional node field in melanoma patients with a minimally invasive procedure.1 Within 3 years of the publication describing the procedure, the accuracy of SLN biopsy was confirmed in two further studies in which the SLN status was found to accurately reflect the status of the entire lymph node field.2,3 The SLN biopsy technique has since become widely accepted as a method of staging the disease of patients with melanoma who have clinically negative lymph nodes. The status of the SLN is used to determine whether further surgical or adjuvant therapy is appropriate, and to assess patient prognosis. The tumor-harboring status of the SLN is the single most important prognostic factor for melanoma patients, surpassing Breslow thickness, ulceration, and mitotic rate.4
To provide accurate prognostic information and to guide appropriate management, it is imperative that all “true” SLNs are accurately identified, excised, and assessed for their tumor-harboring status. In a number of cases, a false-negative result may occur, whereby a patient with disease originally assessed as SLN-negative subsequently develops recurrence in a regional node field. It is disturbing to note that false-negative rates of up to 24.8% have been reported from some major melanoma treatment centers and cooperative groups.5–16 Available evidence suggests that these failures occur not because the SLN biopsy concept is flawed, but because of methodological and technical shortcomings in nuclear medicine, surgery, and histopathology.
In an attempt to identify how many of these false-negative cases might have been because of surgical failures—that is, the removal of a lymph node that was not the SLN identified by the preoperative lymphoscintigraphy—we have developed a technique for assaying antimony in tissue sections. This work is based on the concept that technetium 99 m–labeled antimony trisulfide colloid (99 mTc-Sb2S3) particles used for preoperative lymphoscintigraphy in Australia are retained in the SLN but are absent or present in only tiny amounts in non-SLNs. It has been shown that antimony is preferentially retained by the SLN and can be measured in nodal tissue sections by inductively coupled plasma–mass spectrometry (ICP-MS).17 It has also been demonstrated that human tissue contains only negligible amounts of antimony.17 ICP-MS enables validation of the SLN biopsy procedure26 and can identify false-negative results attributable to inaccurate SLN removal.18
Although the morbidity associated with SLN biopsy is low,11 it is an invasive procedure, with a definite risk of complications and morbidity.19,20 In addition, the financial implications of SLN biopsy must be considered. One estimate of the cost of performing a SLN biopsy procedure on an outpatient basis was US$12,19321—a high cost for providing melanoma treatment services to the community. The development of a minimally invasive technique for the identification of SLN metastases may minimize or eliminate the morbidity of the procedure and provide a potential cost benefit. It should be possible to identify SLNs by preoperative lymphoscintigraphy, localize the SLNs with ultrasound, and then perform a percutaneous fine-needle biopsy (FNB) under ultrasound control.22 Preliminary studies of proton magnetic resonance spectroscopy (MRS) analysis of FNB samples from lymph nodes indicate that this technique has the capacity to distinguish nodes containing metastatic melanoma from uninvolved nodes with high sensitivity, specificity, and accuracy.22,23 In addition to accurately determining the tumor-harboring status of the lymph node by this technique, it is also valuable to verify that the sampled lymph node is a “true” SLN, particularly if it has been assessed as being free of metastatic tumor.
The objective of this study was to develop a method for confirming that a FNB specimen was obtained from a true SLN by measuring the level of antimony (present in radiocolloid used during preoperative lymphoscintigraphy) in the FNB sample by ICP-MS.
Materials and Methods
Instrumentation
All measurements were made with an Agilent 7500ce Inductively Coupled Plasma Mass Spectrometer equipped with a MicroMist glass concentric nebulizer, a Quartz-Scott spray chamber (Peltier cooled, 2°C), and an Agilent three-channel peristaltic pump. The operating conditions were optimized daily to ensure maximum sensitivity. Typical operating conditions are summarized in Table 1. The mass spectrometer was operated in spectrum mode with an integration time of 300 ms on each of the following isotopes: 121Sb, 123Sb, 103Ru (internal standard/control).
TABLE 1.Operating parameters for Agilent 7500ce Inductively Coupled Plasma–Mass SpectrometryParameterConditionRadiofrequency power1500 WSample depth8 mmPlasma gas flow 15.0 L min−1Carrier gas flow1.05 L min−1Peristaltic pump.15 rpsSample read delay60 sRinsing time45 sDwell time100 msReplicates6rps, revolution per second.
Reagents and Chemicals
All reagents used were of the highest purity available. Seventy percent double-distilled nitric acid, 37% hydrochloric acid (Arastar), and 30% hydrogen peroxide were obtained from Sigma-Aldrich, Australia. Ultra-high-purity water was produced by passing distilled water through a Milli-Q deionizing system (Millipore, Australia).
Standards and Certified Reference Materials
Certified reference material GBW 07601 Human Hair Powder (Langfang, China) was selected because of its certified antimony levels. Multiple samples of the reference material were also analyzed with each batch of samples and during method development to ensure the accuracy and precision of the analytic technique.
SLN Biopsy Procedure
Preoperative lymphoscintigraphy was performed to identify the node fields receiving direct lymphatic drainage. This process involved intradermal injections of 99 mTc-Sb2S3 around the primary cutaneous melanoma site, followed by early and delayed imaging with a scintillation camera.24 The location of the SLNs was marked on the overlying skin by the nuclear medicine physician to assist the surgeon in locating SLNs during surgery.
The SLN biopsy procedure was performed within 24 hours of the radiocolloid injection, so that residual radioactivity in lymph nodes could be measured intraoperatively with a handheld gamma probe (Navigator GPS, RMD Instruments, Watertown, MA). Fifteen minutes before the operative procedure, multiple intradermal injections of Patent Blue V dye (Guerbet, Roissy, France) were made around the primary cutaneous melanoma site. SLN identification was based on visualization of the nodal blue dye staining and results from the preoperative lymphoscintigraphy, with gamma probe confirmation. The experimental protocols of this study were approved by the University of Sydney Ethics Review Board in accordance with the precepts established by the Declaration of Helsinki.
FNB Samples
The FNB collection process involved puncturing the fresh SLN specimen within 30 minutes of its surgical removal with a 25-gauge needle attached to a 3-mL plastic syringe. Multiple passes were then made through each quarter of the specimen. A total of 47 FNB samples (from 32 presumptive SLNs and 15 nodes considered to be non-SLNs) were collected. All samples were placed in polypropylene vials containing 300 μL of phosphate-buffered saline (.27 mM of KCl, 13.69 mM of NaCl, 1.52 mM of KH2PO4; pH 7.2) made up in perdeuterated water (phosphate-buffered saline–D2O) and immediately snap-frozen in liquid nitrogen and stored at −70°C.22
Microwave Digestion Procedure
Each FNB sample was transferred to a polypropylene tube and prepared by the previously described microwave digestion procedure.17 Briefly, the sample was digested in a solution containing 300 μL of nitric acid, 300 μL of hydrochloric acid, and 500 μL of hydrogen peroxide. Each sample was digested five times in a 500 W microwave oven on the defrost setting for 30 seconds each time. The digest was then quantitatively transferred to a second polypropylene tube, made up to 10 g with a 1% nitric acid solution, and assayed by ICP-MS.
Statistical Analysis
The unpaired t-test was used to analyze differences between the levels of antimony in the FNB digests from SLNs and non-SLNs. A P value of less than .05 was considered statistically significant.
Results
The certified concentration of antimony in GBW 07601 is .095 ± .012 μg/g (mean ± SD). Replicate analyses of reference materials during method validation and sample analysis gave a mean value of .088 ± .007 μg/g and a relative standard deviation of <9%, confirming the accuracy and precision of the method. The limit of detection (LOD) was evaluated by the 3σ criterion (the LOD is given by mb + 3σb, where mb is the blank measurement mean and σb is standard deviation of five blank measurements) and found to be .048 parts per billion (ppb).
The matrix-matched calibration standards were in the range of 0 to 20 ppb and gave an R2 value of >.9998 during method validation and sample analysis. The relative standard deviation of the slopes of the calibration curves was found to be <10%.
The concentration of antimony in the digested SLNs and non-SLNs was measured, and the results are presented in Tables 2 and 3, respectively. The mean and median concentrations of antimony were .898 and .451 ppb, respectively, in the SLNs (range, <LOD−3.248) and .0145 and .0068 ppb in the non-SLNs (range, <LOD−.1159). These results indicate that the levels of antimony in FNBs from SLNs were significantly greater than from non-SLNs (P < .00005) (Fig. 1).
TABLE 2.Concentration of antimony (ppb) in fine-needle biopsy sample digests collected from sentinel lymph nodesSample No.Antimony concentration (ppb)1<LOD2<LOD3<LOD4<LOD5.06666.09797.17388.29859.302010.308511.325712.380713.381214.386415.390316.404517.497118.607919.640720.724321.8582221.091231.438241.444251.724261.760271.847282.013292.150302.298312.756323.248LOD, limit of detection (.048 ppb).TABLE 3.Concentration of antimony (ppb) in fine-needle biopsy sample digests collected from nonsentinel lymph nodesSample No.Antimony concentration (ppb)1<LOD2<LOD3<LOD4<LOD5<LOD6<LOD7<LOD8<LOD9<LOD10<LOD11<LOD12<LOD13<LOD14<LOD15.1159LOD, limit of detection (.048 ppb).Fig. 1.Antimony concentration in digested fine-needle biopsy samples from sentinel lymph nodes and nonsentinel lymph nodes.
Discussion
Current standard clinical management of patients with primary cutaneous melanoma includes wide local excision of the primary tumor and SLN biopsy for patients considered to be at high risk of having regional node field metastases.25 Technical failures of the procedure could be attributable to errors in lymphoscintigraphy, sentinel lymphadenectomy, or histologic evaluation. In such cases, the potential for disease recurrence in a previously mapped lymph node basin exists.6,8–10,12,14
A reliable technique to confirm the identity of a SLN has the potential to reduce false-negative results attributable to surgical errors. In a previous study, we demonstrated that the analysis of antimony in sectioned nodal tissue could be used to distinguish SLNs and non-SLNs removed from the same nodal basin.26 This technique can be used within the bounds of currently used histopathologic protocols and has the advantage of being applicable to archival paraffin-embedded tissue.
It has also been established that false-negative results may be caused by the removal of a lymph node incorrectly classified as a sentinel lymph node.18 Because neither blue dye nor radioisotope persist after procedures required for microscopic examination, they cannot be used retrospectively to confirm that the “true” SLN had been removed. The only other reported method to confirm SLN identity involves the injection, along with the blue dye, of carbon particles that are retained by the node.27,28 However, these large, dense carbon particles may hinder optimal histologic examination by obscuring metastatic melanoma cells. As far as we are aware, this method is not in routine clinical use.
FNB digests from four nodes presumed to be SLNs (13%) were found to contain extremely low levels of antimony, below the LOD. In a previous study, we also identified SLNs that contained extremely low levels of antimony.26 The most likely reason for the low antimony levels would be inaccurate classification of the node that had been removed as a SLN, when it is in fact a non-SLNs. Other possible explanations to account for the low antimony levels include variable distribution of antimony throughout the node, limited migration of the colloid to the lymph nodes, or tumor deposits preventing colloid uptake. Haigh et al.27 investigated the distribution of carbon dye in SLNs and found a high concentration of carbon particles around the point of entry of afferent lymphatic channels. It is likely that antimony exhibits an analogous distribution pattern. If so, FNB samples that fail to include this region of the SLN may contain falsely low antimony levels. In view of these factors, it would be useful to set a criterion standard for classifying a given node as sentinel or not on the basis of antimony levels assessed by ICP-MS.
However, defining such a criterion on the basis of our small sample set is difficult. In a previous study, we assessed antimony levels in archival tissue sections of paired tumor-positive SLNs and tumor-negative non-SLNs removed from the same regional node field during the same operative procedure from individual patients.26 The aims of this study were to determine whether antimony concentrations could be used to confirm whether removed SLNs were “true” SLNs and to differentiate SLNs from non-SLNs. The median concentration of antimony in the SLNs was .526 ppb and in non-SLNs was .043 ppb (P = .004). By using a cutoff point of .18 ppb (the median concentration of antimony in all SLNs and non-SLNs) to differentiate SLNs from non-SLNs, 20 of 24 SLNs and 20 of 24 non-SLNs were correctly identified by the SLN biopsy procedure.
Although SLN biopsy is a highly accurate method for staging regional lymph nodes, it is an invasive surgical procedure that is costly and is associated with an inherent risk of complication and morbidity.19,20 Pathologic assessment of SLNs is laborious, time-consuming, and costly, and involves examination of multiple hematoxylin and eosin–stained histopathologic sections as well as sections stained with immunohistochemical techniques. The development of a rapid nonsurgical technique that allows detection of metastatic tumor deposits would be of great benefit. It would eliminate the need for surgical excision of lymph nodes and reduce the complexity of pathologic assessment of SLNs. However, to ensure that potential false-negative results are minimized, it is imperative to ensure the accuracy of SLN identification.
Several studies have identified proton MRS as a candidate for the nonsurgical assessment of lymph nodes.22,23 The technique monitors changes in the chemical composition of cells during tumor development and can identify differences that are not morphologically discernible. In these studies, the spectra collected from SLN FNB samples harboring tumor cells contain choline and taurine peaks, which are absent in disease-free samples.23 Another sensitive technique for the detection of melanoma metastases in SLNs is the assessment of tyrosinase mRNA by reverse transcriptase–polymerase chain reaction (RT-PCR).29,30 However, as far as we are aware, RT-PCR assessment of tyrosinase mRNA has not been studied in FNB samples of SLNs. A major potential problem of RT-PCR analysis of FNB samples of SLNs is the issue of false-positive and false-negative results in such specimens and this would need to be assessed before the technique could be used in clinical practice.
The results of our preliminary work provide evidence that determining antimony levels in FNB specimens by ICP-MS can confirm SLN identity and can differentiate SLNs from non-SLNs. This technique may be a useful adjunct to other techniques assessing tumor-harboring status of SLNs on FNB specimens such as MRS. Potentially, proton MRS analysis of a FNB from a node confirmed to be a true SLN by ICP-MS could provide a reliable method for determining the tumor-harboring status of SLNs. Although the results are promising and raise the possibility that SLN assessment may be performed on FNB specimens (rather than histologic specimens of excised SLNs) in the future, further validation studies are necessary before such techniques are used in widespread clinical practice. Furthermore, the highly specialized and expensive nature of the equipment used for ICP-MS will likely limit the technique to specialist centers.

sentinel lymph node
fine-needle biopsy
melanoma
clinical
pathology


Platelet Function in Acute Experimental Pancreatitis

Acute pancreatitis (AP) is characterized by disturbances of pancreatic microcirculation. It remains unclear whether platelets contribute to these perfusion disturbances. The aim of our study was to investigate platelet activation and function in experimental AP. Acute pancreatitis was induced in rats: (1) control (n = 18; Ringer’s solution), (2) mild AP (n = 18; cerulein), and (3) severe AP (n = 18; glycodeoxycholic acid (GDOC) + cerulein). After 12 h, intravital microscopy was performed. Rhodamine-stained platelets were used to investigate velocity and endothelial adhesion in capillaries and venules. In addition, erythrocyte velocity and leukocyte adhesion were evaluated. Serum amylase, thromboxane A2, and histology were evaluated after 24 h in additional animals of each group. Results showed that 24 h after cerulein application, histology exhibited a mild AP, whereas GDOC induced severe necrotizing AP. Intravital microscopy showed significantly more platelet–endothelium interaction, reduced erythrocyte velocity, and increased leukocyte adherence in animals with AP compared to control animals. Thromboxane levels were significantly elevated in all AP animals and correlated with the extent of platelet activation and severity of AP. In conclusion, platelet activation plays an important role in acute, especially necrotizing, pancreatitis. Mainly temporary platelet–endothelium interaction is observed during mild AP, whereas severe AP is characterized by firm adhesion with consecutive coagulatory activation and perfusion failure.
Introduction
Acute pancreatitis (AP) is characterized by an inflammatory affection of the exocrine pancreatic tissue and disturbances of pancreatic microcirculation.1 Depending on the severity of AP, irreversible perfusion failure with consecutive tissue hypoxia and necrosis can complicate the course of the disease and trigger systemic inflammatory and septic complications.2 The pathophysiology of AP has been investigated with regard to microcirculatory changes in several studies.1–5 Attention was paid especially to erythrocyte flow patterns, leukocyte–endothelium interaction, and rheological approaches to improve perfusion and inhibit irreversible tissue damage.1,3–5 Leukocyte–endothelium interaction as an early event of the inflammatory response has been characterized as a key step in the pathophysiology of AP.6 Besides, activation of the humoral coagulation cascade plays an important role in the development of microcirculatory disorders in AP.7,8 However, the role of platelets as the cellular elements of hemostasis that can functionally link inflammatory cells and humoral coagulation factors has not been investigated.
The aim of this study was to investigate platelet activation and function in experimental AP.
Materials and Methods
Animals The experiments were performed in 54 male Wistar rats weighing 270 to 335 g. Animals were fasted overnight with free access to water before the experiments. Care was provided in accordance with the guidelines published in the “Guide for Care and Use of Laboratory Animals” (National Institutes of Health, publication no. 85-23, 1985). Surgical anesthesia was induced with intraperitoneal injection of pentobarbital (25 mg/kg) and intramuscular injection of ketamine (40 mg/kg) for the procedures of catheter placement and induction of pancreatitis. Anesthesia during intravital microscopy was induced by intravenous injection of pentobarbital (10 mg/kg). Polyethylene catheters (inner diameter 0.5 mm) were placed in the right jugular vein and left carotid artery, tunneled subcutaneously to the suprascapular area, and brought out through a steel tether that allowed the animals’ free movement and access to water during the experiments.
Monitoring blood samples Mean arterial pressure and heart rate were monitored during intravital microscopy by an electromechanical pressure transducer (Baxter Uniflow, Baxter Healthcare Cooperation, Deerfield, IL, USA). Arterial blood samples for determination of serum amylase were obtained before (baseline) and 24 h after (end point) pancreatitis was induced. Serum amylase was determined by standard laboratory methods (Hitachi automatic analyzer, Boehringer Mannheim, Germany).
Animal models Animals were divided into three groups. In each group, pancreatic microcirculation was evaluated in 12 animals by intravital microscopy, and morphological changes were assessed in six animals by histology. In the control group, animals underwent sham operation and received Ringer’s solution only. Acute pancreatitis of graded severity was induced in the other groups either as mild or severe AP. Mild AP was induced by intraarterial infusion of cerulein (5 μg kg−1 h−1) for over 6 h. Cerulein was reconstituted in saline solution, and infusion volume was 4 ml/kg/h. Severe necrotizing pancreatitis was induced by infusion of bile salt (glycodeoxycholic acid [GDOC] 2.5 mM/l) into the pancreatic duct in combination with intraarterial infusion of cerulein (5 μg kg−1 h−1) for over 6 h as described by Schmidt et al.9 in detail. Bile-salt infusion into the pancreatic duct was performed in a volume- (1.2 ml/kg), time- (5 min), and pressure- (30 mmHg) controlled manner.In each of the models, animals received saline solution during the observation period (0.9%, 4 ml kg−1 h−1). Intravital microscopy was performed 12 h after the induction of pancreatic injury, and histological changes and blood samples were assessed 24 h after the infusions were started.
Platelet preparation One milliliter of whole blood was withdrawn before intravital microscopy. Platelets were separated and stained according to the method originally described by Massberg et al.10 Briefly, platelets were stained by rhodamine 6G and separated by 2 cycles of centrifugation under the addition of prostacyclin. After suspending and washing the separated platelets, blood cell count was performed to calculate the number of platelets per microliter and to rule out animal-specific differences in the number of platelets. Platelets were then reinjected, and intravital microscopy was performed.
Intravital microscopy The abdomen was reopened, and the pancreas was carefully exteriorized in a horizontal position through the midline incision after the animal was placed on the right side. The duodenal loop with the head of the pancreas was carefully fixed on an anatomically designed stage in a temperature-controlled (37°C) Ringer’s bath. Afterward, intravital microscopy was performed as described below. The animals were killed after the completion of intravital microscopy by a pentobarbital overdose.
Erythrocyte and leukocyte assessment A 0.5 ml/kg of erythrocytes (hematocrit 50%) labeled with fluorescein isothiocyanate (FITC) as described before11 was applied intravenously. In addition, 1 ml/kg of rhodamine-6G solution was applied intravenously to label leukocytes in vivo.12 Intravital microscopy was performed after an equilibration period of 15 min using a fluorescent microscope (Leitz, Wetzlar, Germany) with a 20-fold water immersion objective. An epi-illuminescent xenon lamp with an excitation filter of 450–490 nm was used for visualization of FITC-labeled erythrocytes and an excitation filter of 540–630 nm for rhodamine-labeled leukocytes.
Platelet assessment After platelet reinjection, intravital microscopy was performed by an epi-illuminescent xenon lamp with an excitation filter of 540–630 nm.
Off-line analysis Images were transferred to a monitor and simultaneously recorded on a videotape recorder. In each animal, five capillary fields of the exocrine pancreas and five postcapillary venules (20–40 μm) were recorded for 1 min. Off-line analysis was performed using a specially designed computer program (Capimage, Dr. Zeintl, Heidelberg, Germany). Erythrocyte velocity and platelet velocity were determined for 10 cells in each capillary field and venule. Additionally, temporarily (rolling) and permanently (sticking) adherent leukocytes and platelets were determined in pancreatic venules and capillary fields. Rolling cells were defined as cells with less than 66% of red blood cell velocity, whereas sticking cells are those that were adherent to the vessel wall for the whole observation period.13
Edema A portion of pancreatic tissue was trimmed of fat and weighed. Pancreatic water content was determined by the ratio of the initial weight (wet weight) of the pancreas to its weight after incubation at 60°C for 72 h (dry weight).
Histology The pancreas was immediately removed after killing and was fixed in 4% buffered formalin solution. It was then embedded in paraffin, cut, and stained with hematoxylin eosin. Histopathological evaluation was performed in a blinded fashion. For quantification of edema, inflammation, and necrosis, a modification of the scoring system originally described by Schmidt et al.9 was used, ranging from 0 to 3 (no pathological changes to severe injury).
Assessment of thromboxane A2 Thromboxane A2 was measured in frozen serum by commercially available enzyme-linked immunosorbent assay (University of Freiburg, Germany).
Statistical analysis Results are shown as mean ± SEM. Student’s t test was used when the data had a normal distribution, whereas Kruskal–Wallis and Mann–Whitney tests were utilized when the distribution was not normal. Statistical significance was accepted at the 5% level (p < 0.05).
Results
Serum amylase Twelve hours after the induction of AP, serum amylase increased significantly compared to control animals. Hyperamylasemia was comparable in both mild and severe AP indicating the presence of pancreatic cell damage. However, amylase was not a marker for the extent of tissue damage or disease severity (Table 1).
Table 1Serum Parameters, Wet–Dry Ratio, and Histopathology ControlMild APSevere APSerum parametersAmylase (U/l)586 ± 11627,200 ± 4,012*27,317 ± 3,220*Thromboxane A2 [pg/50 μl]15.3 ± 10.347.8 ± 12.1*61.9 ± 15.8*Wet–dry ratio2.87 ± 0.796.96 ± 0.95*4.77 ± 0.70HistopathologyInflammation0.25 ± 0.421.31 ± 0.08*1.95 ± 0.17*†Necrosis0.08 ± 0.201.10 ± 0.11*1.70 ± 0.23*†*p < 0.05 vs control group†p < 0.05 vs mild acute pancreatitis
Serum thromboxane A2 Thromboxane A2, as a marker of platelet activation, showed significantly higher levels in both AP groups compared to control animals after 24 h. Thromboxane liberation correlated with severity of AP, with the highest levels being present in animals with necrotizing AP (Table 1).
Intravital microscopy Erythrocyte velocity decreased significantly in mild as well as severe AP in both capillaries and venules compared to control animals. Platelets showed comparable flow features. Flow velocity decreased under both AP conditions, with a highly significant decrease in severe AP in venules and capillaries (Table 2). These changes were paralleled by increased interaction between leukocytes and endothelium (Table 2). Platelet adhesion in capillaries and venules increased significantly in both mild and severe AP (Figs. 1 and 2). Reversible adhesion (rolling platelets) were comparable during both forms of AP, whereas the increase in irreversible adhesion (sticking platelets) depended on the severity of AP and showed peak platelet–endothelium adherence in necrotizing AP (Figs. 1 and 2).
Table 2Results of the Intravital MicroscopyIntravital microscopyControlMild APSevere APErythrocyte velocity (capillary) (mm/s)0.65/0.020.42/0.01*0.36/0.01*Erythrocyte velocity (venule) (mm/s)0.93/0.110.77/0.170.58/0.10*†Platelet velocity (capillary) (mm/s)0.54 ± 0.040.35 ± 0.03*0.29 ± 0.03*Platelet velocity (venule) (mm/s)0.67 ± 0.050.63 ± 0.020.53 ± 0.05*Rolling leukocytes (capillary)1.3 ± 0.24.5 ± 1.4*9.0 ± 1.7*†Rolling leukocytes (venule)1.3 ± 0.214.8 ± 1.2*18.9 ± 1.9*Sticking leukocytes (capillary)1.1 ± 0.310.2 ± 1.8*7.2 ± 0.7*Sticking leukocytes (venule)0.7 ± 0.15.6 ± 0.9*13.5 ± 2.0*†*p < 0.05 vs control group†p < 0.05 vs mild acute pancreatitisFigure 1Intravital microscopy, capillary platelet adhesion (one per field). Control group (gray), mild acute pancreatitis (white), and severe acute pancreatitis (striped). Reversible platelet adhesion in mild and severe acute pancreatitis (left columns); irreversible platelet adhesion (right columns). *p < 0.05 vs control group, †p < 0.05 vs mild acute pancreatitis.Figure 2Intravital microscopy, venular platelet adhesion (one per 100 μm). Control group (gray), mild acute pancreatitis (white), and severe acute pancreatitis (striped). Reversible platelet adhesion in mild and severe acute pancreatitis (left columns); irreversible platelet adhesion (right columns). *p < 0.05 vs control group, †p < 0.05 vs mild acute pancreatitis.
Tissue edema (wet/dry ratio) Supramaximal cerulein stimulation induced a significant increase in pancreatic water content compared to control animals. In contrast, there was only a slight increase in tissue edema after GDOC treatment (Table 1).
Histopathology Control animals showed no histopathological changes after sham operation and 24 h infusion therapy. Histopathology of mild AP was characterized by significant edema formation, inflammatory tissue infiltration, and acinar cell necrosis. In severe AP, the changes regarding inflammation and necrosis were significantly more pronounced (Table 1).
Discussion
In the present study, we have investigated platelet function in experimental models of AP. We chose two animal models to induce a mild edematous or a severe necrotizing course of AP. Both models are established, well characterized, and have been used in numerous studies.9,14,15 The induction of AP in these models results in a standardized grade of tissue damage, either mild or severe, with very little variance within each group. Therefore, the use of these models allows us to rule out the significant influence of preparatory or other methodological problems on the comparability of the results.
Analysis of platelet function by intravital microscopy has been established and standardized for examination of liver and small bowel perfusion by Massberg et al.10 We have modified this method to investigate the pancreas.15 In the present study, we could demonstrate that this method is not only suitable for the examination of healthy pancreas but also for the detailed analysis of pancreatic microcirculation in mild and severe AP.
Acute pancreatitis is characterized by an impairment of microcirculation due to an activation of inflammatory cells with a consecutive increase of leukocyte–endothelium interaction. These pathophysiological events mediate an inflammatory tissue infiltration, edema, and hemorrhagic lesions. While the inflammatory response is well investigated, the platelet function and the role of the coagulation cascade have not yet been investigated in detail.
It is well known that the inhibition of certain coagulatory steps, e.g., by applying hemodiluting or anticoagulatory substances, improves the outcome of AP.16,17 Coagulation and hemostasis comprise two interacting pathways: humoral coagulatory factors leading to the activation of fibrinogen as the final step of the coagulation cascade and cellular factors, which are represented by activated platelets. Different mechanisms of platelet interaction are responsible for their physiological function, namely, interactions with endothelium, leukocytes, and humoral coagulatory and inflammatory proteins.18,19
In the present study, we could demonstrate that the platelet–endothelium interaction increases during AP and correlates with the degree of its severity. Comparable to leukocyte–endothelium interaction, temporary and permanent adhesions of platelets to the vessel wall were evident in our experiments. This correlates with the activation patterns that have been observed in vivo in ischemia models of the liver and the pancreas,15,20 as well as in vitro.21 Therefore, it seems likely that these activation patterns reflect the severity of the pancreatic affection, leading to reversible adhesion in mild AP and irreversible adhesion in more severe organ affection. Especially, the firm adhesion of platelets contributes to microcirculatory disturbances and may induce perfusion failure and tissue necrosis in the progression to severe AP. The significantly elevated thromboxane levels correlate well with platelet activation and microcirculatory failure observed during intravital microscopy. The increase in serum thromboxane elucidates one mechanism of our results as it executes a direct platelet stimulation and leads to the conversion of “resting” to “activated” platelets with the consecutive adhesive action. Furthermore, thromboxane does not only activate platelets but also acts as a complex pathophysiological mediator with multiple other targets. Its effects include leukocyte activation, upregulation of proinflammatory cytokines, and strong vasoconstricting effects. These are mediated via phosphatidylcholine and phosphatidylcholine-specific phospholipase-C pathway leading to a tonic contraction in smooth muscles and upregulating other vasoactive substances.22,23 Especially, this vasoconstrictor mechanism may additionally contribute to perfusion failure in the course of AP as observed in our study. How far platelet inhibition itself could be an approach to attenuate the course of AP experimentally or clinically is hypothetical but should certainly be addressed to in further studies. Possible aims could be adhesion molecules such as selectins or platelet receptors and also synthesis of thromboxane and prostaglandins.
Platelet activation was accompanied by leukocyte activation in the present study. An interaction between these two cell types has been demonstrated by the different authors in the past.24–26 Among others, P-selectin seems to be one of the most important adhesion molecules, which links the inflammatory and procoagulatory cascades and has the potency to activate leukocytes and platelets as the cellular elements of either pathway.18,19,25,26 Besides their adherence to endothelial cells, activated platelets form stable aggregates with leukocytes. This results in a combined inflammatory and coagulatory contribution to thrombus formation and is also mediated by P-selectin and beta-integrins.27–29 Especially, the formation of microthrombotic vessel occlusion with microcirculatory perfusion failure and consequent ischemia, hypoxia, and tissue necrosis was reflected by the intravital microscopic results in the present study.
Conclusion
The results of the present study show that activation and adhesion of platelets play an important role during AP. Platelet–endothelium and platelet–leukocyte interactions as well as thromboxane liberation show a correlation with the severity of experimental AP and seem to be of distinct importance in the progression from mild to severe necrotizing AP. A possible therapeutic use of these pathophysiological events should be evaluated in further studies.

platelets
acute pancreatitis
microcirculation
leukocytes
coagulation
endothelium interaction


Optical spectroscopic studies of light-harvesting by pigment-reconstituted peridinin-chlorophyll-proteins at cryogenic temperatures

Low temperature, steady-state, optical spectroscopic methods were used to study the spectral features of peridinin-chlorophyll-protein (PCP) complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). Absorption spectra taken at 10 K provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment–protein interactions responsible for the positions of the Qy bands of the chlorophylls. The study reveals that the functional groups attached to Ring I of the two protein-bound chlorophylls modulate the Qy and Soret transition energies. Fluorescence excitation spectra were used to compute energy transfer efficiencies of the various complexes at room temperature and these were correlated with previously reported ultrafast, time-resolved optical spectroscopic dynamics data. The results illustrate the robust nature and value of the PCP complex, which maintains a high efficiency of antenna function even in the presence of non-native chlorophyll species, as an effective tool for elucidating the molecular details of photosynthetic light-harvesting.
Introduction
Photosynthetic organisms have bioengineered a wide variety of protein-based structures to carry out photosynthetic light-harvesting (Frigaard et al. 2004). High-resolution molecular structures of many of these systems are providing important clues into the molecular features that control light-harvesting in photosynthesis (Germeroth et al. 1993; Hofmann et al. 1996; Hu et al. 1995; Koepke et al. 1996; Kühlbrandt et al. 1994; Law et al. 2004; Liu et al. 2004; McDermott et al. 1995; Roszak et al. 2003). To understand fully how these naturally occurring pigment–protein complexes harvest light so efficiently, it is necessary to employ spectroscopic tools that reveal directly the dynamics and efficiency of energy transfer and the nature of the excited energy states associated with the bound pigments. The spectroscopic studies are aided by the ability to make systematic alterations in the structures of the light-harvesting complexes, and then to examine how these changes manifest themselves in the spectroscopic observables, dynamics of energy transfer and efficiency of antenna function (Akahane et al. 2004; Cammarata et al. 1990; Chadwick et al. 1987; Crielaard et al. 1994; Croce et al. 1999; Davidson and Cogdell 1981; Frank 1999; Fraser et al. 1999; Morosinotto et al. 2002; Olivera et al. 1994; Olsen et al. 1997; Plumley and Schmidt 1987; Remelli et al. 1999; Struck and Scheer 1991; Struck et al. 1992).
The peridinin-chlorophyll-protein (PCP) complex from the dinoflagellae, Amphidinium carterae, is an example where many of these approaches have been applied. Atomic resolution structural analysis by X-ray diffraction to 2.0 Å resolution (Hofmann et al. 1996) has been augmented by pigment reconstitutions and protein refolding studies (Miller et al. 2005; Polívka et al. 2005). Steady-state and time-resolved spectroscopy (Bautista et al. 1999a; Kleima et al. 2000a; Kleima et al. 2000b; Krueger et al. 2001; Linden et al. 2004; Shima et al. 2003; Zigmantas et al. 2002; Zimmermann et al. 2002) has been carried out on the excited singlet and triplet states associated with the bound pigments. The X-ray crystallographic analysis of PCP revealed a trimeric structure of subunits (Hofmann et al. 1996) where eight peridinins and two chlorophyll a (Chl a) molecules (Fig. 1) bind non-covalently in a roughly C2 symmetrical arrangement in each of the three individual subunits. The peridinins are assembled in clusters of four within the subunits, surrounding, and in van der Waals contact with, one chlorophyll a (Chl a) (Fig. 1). The two Chl molecules within a subunit have a center-to-center distance of 17.4 Å. Steady-state and ultrafast optical spectroscopic investigations on peridinin in solution and on the main-form PCP (MFPCP) have explored its photoinduced excited-state processes and energy transfer pathways (Carbonera et al. 1999; Damjanovic et al. 2000; Kleima et al. 2000; Krueger et al. 2001; Ritz et al. 2000; Shima et al. 2003; Zimmermann et al. 2002). In addition, a variant form, the high-salt PCP (HSPCP), has been studied in the same manner (Ilagan et al. 2004; Sharples et al. 1996).
Fig. 1Structure of the pigments in a monomeric subunit of the MFPCP complex. The coordinates were taken from Protein Data Bank with 1PPR code. Also shown are the structures of peridinin and the chlorophylls used in this study
Adding to the arsenal of PCP complexes that can be used for structural and spectroscopic studies, Miller and coworkers (Miller et al. 2005) prepared and Polívka and coworkers (Polívka et al. 2005) studied the N-domain and full-length PCP apoproteins expressed in Escherichia coli and reconstituted with the total pigment extract from native PCP to produce a fully functional complex with a high peridinin-to-Chl a energy transfer efficiency. Also, they demonstrated that in the N-domain PCP apoprotein the bound Chl a molecules could be replaced by other Chls using the N-domain apoprotein incorporating the requisite stoichiometric amount of peridinin.
In this paper, we present a low temperature, steady-state, optical spectroscopic investigation of the spectral features of PCP complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). We use fluorescence excitation spectroscopy to examine in detail the peridinin-to-Chl energy transfer efficiencies of the various complexes at room temperature and correlate the results with previously reported ultrafast, time-resolved optical spectroscopic dynamics data (Polívka et al. 2005). The low temperature data provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment–protein interactions responsible for the observed Chl transition energies.
Materials and methods
Sample preparation
The PCP complexes were reconstituted from N-domain apoprotein with peridinin and either Chl a, Chl b, Chl d, 3-acetyl-Chl a or BChl a (Fig. 1) as described (Miller et al. 2005). Size exclusion chromatography shows the reconstituted PCP to be approximately 40 kDa in apparent molecular mass; i.e., the same as that of the native PCP, suggesting that the samples used here consist of monomeric protein structures. The purified reconstituted PCP complexes were stored at 4°C until ready for use in the spectroscopic experiments. For the cryogenic experiments, the PCP complexes were dissolved in a buffer containing 50 mM Tricine, 20 mM KCl buffer and 60% (v/v) glycerol at pH 7.6.
Chl a and Chl b were obtained from Sigma-Aldrich. Chl d and BChl a were extracted from the cyanobacterium, Acaryochloris marina, and the bacterium, Rhodospirillum rubrum, respectively. Total pigments were extracted from A. marina cells with 2-butanol and the extract dried in a gentle stream of nitrogen and Chl d was purified by HPLC as described below. BChl a was extracted from R. rubrum whole cells with 50:50 (v/v) acetone:methanol. The extracted solution was filtered, dried and redissolved in 50:50 (v/v) petroleum ether:methanol. The methanol phase, in which BChl a was dissolved, was collected and dried with nitrogen gas. The BChl a solution was subjected to an alumina column chromatography with increasing percent of acetone in hexane. 3-Acetyl-Chl a was obtained as an oxidation product of BChl a during its isolation from R. rubrum. All Chls were further purified using a Millipore Waters 600E HPLC system employing a Nova-Pak C18 column. For Chl a and Chl b, isocratic runs of acetone:methanol:water (75:20:5, v/v/v) were used as the mobile phase with a flow rate of 0.5 mL min−1. For Chl d and BChl a, the mobile phase used a combination of solvent A (acetonitrile:methanol—50:50, v/v) and solvent B (solvent A:water—50:50, v/v). The run was programmed as follows: 0–5 min, linear gradient from 80% solvent A and 20% solvent B to 99% solvent A and 1% solvent B; and, 5–30 min, isocratic 99% solvent A and 1% solvent B with a flow rate of 1.0 mL min−1. 3-acetyl-Chl a was separated from BChl a using solvent A (acetonitrile:methanol:water—87:10:3, v/v/v) and solvent B (hexane:propanol—4:1, v/v) as the mobile phase. The mobile phase was programmed as follows: 0–20 min, linear gradient from 100% solvent A to 95% solvent A and 5% solvent B; and, 20–30 min, isocratic 95% solvent A and 5% solvent B with a flow rate of 1.0 mL min−1. The collected samples were dried with a gentle stream of nitrogen gas and then redissolved in 2-methyl tetrahydrofuran (2-MTHF).
Spectroscopy
Absorption spectra were taken at 10 K using a Cary 50 UV-visible spectrometer and a Janis model STVP100 helium vapor flow cryostat using 1 cm pathlength polymethacrylate cuvettes. The fluorescence and fluorescence excitation spectra of the PCP complexes were taken at 77 K using a Jobin Yvon Horiba Fluorolog-3 spectrofluorometer model FL3-22 on samples immersed in a custom built optical dewar (Kontes) as described (Ilagan et al. 2004).
Results
Absorption spectroscopy
The 10 K absorption spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, 3-acetyl-Chl a or BChl a are shown in Fig. 2. All of the complexes exhibit strong absorption in the region 450–575 nm associated with peridinin. For the complexes reconstituted with Chl a and Chl b, a double peak is evident in the Soret region (near 450 nm), but this is not seen in complexes containing Chl d, 3-acetyl-Chl a or BChl a. In the long-wavelength absorption region of the spectra, the Qy band of all the Chls is very sharp and appears at 646 nm for Chl b, 667 nm for Chl a, 682 nm for 3-acetyl-Chl a, 697 nm for Chl d and 782 nm for BChl a. All the Qy bands are blue-shifted compared to the spectra taken at room temperature. Also, except for Chl d that exhibits a weaker shift of 59 ± 6 cm−1, all are shifted by the same amount, 110 ± 15 cm−1. Also, in the PCP complex containing BChl a, a second peak on the long-wavelength side of the primary Qy feature is observed at 799 nm (Fig. 2) and a trace of 3-acetyl-Chl a can be seen in the absorption spectrum of the complex with BChl a.
Fig. 2Absorption spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, aChl a (3-acetyl-Chl a) or BChl a. The spectra were taken at 10 K and were normalized at 500 nm
For comparison and analysis of the Chl spectra of the complexes, absorption spectra of the isolated and purified Chls in 2-MTHF were taken at 10 K. 2-MTHF forms a clear glass at that temperature and has been used previously in this type of analysis (Ilagan et al. 2004). Figure 3 shows the 10 K absorption spectra of the Chl solutions in the Qy region plotted on a wavenumber scale. The spectra were analyzed by Gaussian deconvolution and were well fit using a sum of two components that were allowed to vary in amplitude, position and width built on a broad baseline. The Gaussian deconvolutions of the solution spectra were all very similar (Fig. 3) and revealed a prominent spectral origin and a broad phonon wing at higher energy. The wavelengths of the prominent peaks computed from the wavenumber fits are 670 nm for Chl a, 654 nm for Chl b, 697 nm for Chl d, 689 nm for 3-acetyl-Chl a and 777 nm for BChl a. The results indicate the chlorophyll–protein interaction induces a blue-shift in the case of Chl a, Chl b and 3-acetyl-Chl a, no shift for Chl d and a red-shift for BChl a.
Fig. 3Gaussian deconvolutions of the 10K absorption spectra in the Qy region of purified Chls in 2-MTHF solvent. The fit (dashed line) is the sum of two Gaussian components built on a broad background. The data for the Gaussian deconvolution of the Chl a spectrum was taken from a previous study (Ilagan et al. 2004)
Figure 4 shows the 10 K absorption spectral profiles of the reconstituted PCP complexes in the Qy region on a wavenumber scale fitted using Gaussian functions that were also allowed to vary in amplitude, position and width. The spectra from the PCP complexes containing Chl a, Chl b and BChl a required four Gaussian components on a broad absorption background (Fig. 4A, B, E) for a good fit. The spectra from the complexes having Chl d and 3-acetyl-Chl a required only two Gaussian functions on a broad absorption background (Fig. 4C, D) for a good fit.
Fig. 4Gaussian deconvolutions of the 10K absorption spectra in the Qy region of PCP complexes reconstituted with peridinin and: (A) Chl a; (B) Chl b; (C) Chl d; (D) aChl a (3-acetyl-Chl a); and (E) BChl a
Spectral reconstruction
Linear combinations of the absorption spectra of peridinin and the various Chls taken in 2-MTHF at 10 K were used to reconstruct the spectral line shapes of the complexes in the region 375–600 nm. According to previously published procedures (Ilagan et al. 2004), individual peridinin spectra were constrained to have identical intensities, but were allowed to vary with respect to wavelength. The spectra of the individual Chls were allowed to have any intensity and wavelength. For all of the complexes, two Chl and eight peridinin spectra were used to fit the lineshapes (Fig. 5A–E). The spectra of all the complexes were best fit using two blue-shifted peridinins and three separated pairs of peridinin spectra (Fig. 5A–E), one of which needed to be significantly red-shifted to accommodate the long-wavelength region of the spectra. In the complexes containing Chl a and Chl b, the Soret bands of the two Chls are split and have noticeably different intensities. This required two separate Chl spectra to reproduce the absorption lineshapes in this region (Fig. 5A, B; Table 1). No splitting of the Soret bands was evident for the complexes with Chl d, 3-acetyl-Chl a and BChl a, so the spectra were able to be fit in this region assuming that the two Chls were identical. A summary of the wavelengths of the spectral origins of the individual peridinins and the Chl bands assigned in this analysis is given in Table 1.
Fig. 5Reconstruction of the 10K absorption spectra of the reconstituted PCP complexes in the 375–600nm region. The analysis was carried out by linearly combining individual 10 K absorption spectra of peridinin and Chl taken in 2-MTHF solvent as described in the textTable 1Spectral origins of the eight peridinins and the positions of the Soret peaks and Qy bands of the two Chls derived from the spectral analysis of the 10 K absorption spectra of the reconstituted PCP complexesPCP complexPeridininChlReferenceSoretQy123456781212MFPCP470485522522526526544544434439665667(Ilagan et al. 2004)HSPCPna489533533533533533na436441667676(Ilagan et al. 2004)Chl a472 ± 2486 ± 2510 ± 1510 ± 1529 ± 1529 ± 1548 ± 1548 ± 1434 ± 1440 ± 1667668Chl b470 ± 3485 ± 2508 ± 1508 ± 1527 ± 2527 ± 2546 ± 2546 ± 2458 ± 1461 ± 1644647Chl d474 ± 2490 ± 2513 ± 1513 ± 1530 ± 1530 ± 1546 ± 1546 ± 1456 ± 2456 ± 2697697aChl a467 ± 2482 ± 2511 ± 2511 ± 2530 ± 2530 ± 2543 ± 1543 ± 1446 ± 1446 ± 1683683BChl a472 ± 2486 ± 2507 ± 2507 ± 2531 ± 1531 ± 1545 ± 1545 ± 1365 ± 1365 ± 1782782The numbers are in nm units and the uncertainties represent the range of values that give an acceptable fit to the dataMFPCP is main-form PCP, HSPCP is high-salt PCP, aChl a is 3-acetyl-Chl a, na is not applicable
Fluorescence spectroscopy
Fluorescence spectra of the reconstituted PCP complexes at 77 K are shown in Fig. 6. The spectra display a prominent narrow band at 671 nm (full width at half maximum, FWHM = 8.5 nm) for Chl a, 647 nm (FWHM = 10 nm) for Chl b, 699 nm (FWHM = 8 nm) for Chl d, 686 nm (FWHM = 9.5 nm) for 3-acetyl-Chl a and 787 nm (FWHM = 14.5 nm) for BChl a. All of the spectra show structured vibronic features to the red of the main band. In the PCP complex with BChl a, a shoulder at 798 nm on the red side of the main peak is clearly evident.
Fig. 6Fluorescence spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, aChl a (3-acetyl-Chl a) or BChl a taken at 77 K. The spectra were normalized to their λmax values
Fluorescence excitation spectroscopy
Figure 7 shows the fluorescence excitation spectra of the complexes recorded at room temperature overlaid with their 1-T spectra, where T is transmittance. The Chl fluorescence was monitored at various wavelengths between 675 and 850 nm, and the fluorescence excitation spectra were found to be independent of the monitoring wavelength. The peridinin-to-Chl energy transfer efficiencies obtained from the spectra were averaged over the 450–515 nm wavelength range and were: 94 ± 2% (Chl a), 92 ± 2% (Chl b), 96 ± 3% (Chl d), 99 ± 1% (3-acetyl-Chl a) and ∼100% (BChl a).
Fig. 7Overlay of the room temperature fluorescence excitation (ex) and 1-T (where T is transmittance) spectra of reconstituted PCP complexes containing: (A) Chl a; (B) Chl b; (C) Chl d; (D) aChl a (3-acetyl-Chl a); and (E) BChl a. The intensity of each fluorescence excitation spectrum was normalized to correspond to the intensity of its corresponding 1-T spectrum in the Qy region
Discussion
Low temperature absorption spectra
The 10 K absorption spectra of the reconstituted PCP complexes (Fig. 2) show significantly higher resolution than the spectra taken at room temperature (Polívka et al. 2005). This allows a more detailed look at how changes in the structure of the incorporated Chl affect the spectral profiles. The 10 K lineshape of the PCP complex reconstituted with Chl a (Fig. 2) is very similar to that from the native PCP complex (Ilagan et al. 2004), the major characteristics being the splitting of the Soret band near 435 nm and the shoulder on the red side of the peridinin maximum. These features are clearly evident (Figs. 2, 5A) in both reconstituted and native complexes (Ilagan et al. 2004). This supports the notion that the structures of these two complexes are very similar.
The 10 K absorption spectra of the reconstituted PCP complexes (Fig. 2) also display blue-shifted Qy bands compared with thse spectra of the complexes taken at room temperature, and except for Chl d that exhibits a shift of 59 ± 6 cm−1, are all shifted by 110 ± 15 cm−1. This is opposite to the low temperature-induced red-shift observed for the 850 nm-absorbing BChl molecules in LH2 complexes from photosynthetic bacteria which was explained in terms of a structural change of the LH2 complex upon glass formation at low temperatures that increased excitonic coupling between the BChls (Wu et al. 1997). This cannot be the case for the PCP complexes here because the two Chl molecules are too far apart (17.4 Å) to be excitonically coupled. However, the blue-shift may result from a slight change in the planarity of the Chl macrocycles. Assuming that glass formation induces a structural change (Wu et al. 1997) in the immediate environment of the Chls in the PCP complexes, one may expect a corresponding change in the conformation of the macrocycles. It has been shown that these conformational changes may induce either red- or blue-shifts of Chl Qy bands (Gudowska-Nowak et al. 1990). The fact that the shift is very similar (110 ± 15 cm−1) for all chlorophyll species except Chl d suggests that the structural change affects the macrocycle in the vicinity of Ring I because all the Chls except Chl d have a large substituent in the position R1 (Fig. 1). Thus, if one assumes a low-temperature-induced protein structural change in the immediate environment of Ring I, this may lead to a distortion of the Chl macrocycle and consequently to the observed blue-shift. For Chl d, the smaller aldehyde substituent at position R1 may be less susceptible to protein structural changes and to inducing conformational distortion of the Chl macrocycle.
The Gaussian deconvolutions of the Chl solution spectra in the Qy region (Fig. 3) serve as guides in the analysis of the absorption spectra of the reconstituted PCP complexes. Comparing the spectrum in the Qy region of the Chl a-reconstituted PCP (Fig. 4A) with that of Chl a in solution (Fig. 3A) shows that two distinct pairs of Chl a Gaussian functions built on a broad background are needed to reproduce the spectrum of that complex. Likewise, for the PCP complex containing Chl b, two pairs of Gaussian components having different prominent spectral peaks and zero-phonon lines built on a broad background were needed to reproduce the spectrum of the protein complex (Fig. 4B). However, for the PCP complexes containing Chl d or 3-acetyl-Chl a, only two Gaussian components on a broad background were required to fit the spectra in the Qy band region indicating that Chl d and 3-acetyl-Chl a in these complexes are in highly symmetric environments. This conclusion is supported by the observation that the PCP complexes reconstituted with Chl a and Chl b show a splitting in the Soret band region (Fig. 5A, B), but all the other complexes do not. This indicates a less symmetric environment for the Chls bound in the complexes containing Chl a or b.
The observation that the spectra of the PCP complexes containing Chl a and Chl b show splittings in the Soret and Qy regions attributable to environmental asymmetry, but the spectra from the Chls in the other complexes do not, may also be traceable to the different functional groups on Ring I of the Chls. A view of the published structure of the MFPCP complex focusing on Chl 601 in the vicinity of Ring I shows that this part of the Chl macrocycle is in close proximity (within 4 Å) to peridinins 612 and 614 (Fig. 8). The distance between the vinyl group and the peridinins is closer than to any amino acid residue in the protein. Leucine-204 comes closest at ∼5 Å (Fig. 8). The splitting of the Soret and Qy spectral bands in the Chl a and Chl b PCP complexes implies that the vinyl group on Chl a and Chl b may adopt different configurations leading to distinct interactions between these Chls and the peridinins. In contrast, the carbonyl groups on Ring I of Chl d, 3-acetyl-Chl a and BChl a may have only a single configuration and may also sterically hinder the approach of peridinins 612 and 614 toward the Chl in those complexes, thereby minimizing the interaction between pigments, rendering the Chl spectra equivalent.
Fig. 8A view of the structure of the MFPCP complex focusing on the Ring I of Chl 601. Per 612, Per 614 and Leu 204 are in close proximity to the Ring I of Chl 601. The coordinates of the structure were taken from Protein Data Bank with 1PPR code
The results from the deconvolution of the spectral features for the PCP complex containing BChl a are unusual. In addition to the main Qy absorption band at 782 nm (Figs. 2, 4E), a shoulder at 799 nm is observed which is highly reminiscent of the 4 K absorption spectrum of the antenna BChl a–protein (Fenna–Matthews–Olson, FMO) complex from Prosthecochloris aestuarii (Johnson and Small 1991; Wendling et al. 2002; Whitten et al. 1978). The spectral features in the Qy region are attributed to a combination of site shifts and dipolar interactions among the seven BChl a molecules within one subunit of the FMO protein (Wendling et al. 2002). The crystal structure of this FMO complex has been determined to 2.8 Å resolution (Matthews et al. 1979) and later refined to 1.9 Å resolution (Tronrud et al. 1986). The FMO complex consists of three identical subunits and each subunit contains seven BChl a molecules where the center-to-center distances between BChls vary from 11 to 14 Å (Matthews et al. 1979). This is smaller than the center-to-center distance of 17.4 Å reported from the X-ray crystallographic analysis of the MFPCP complex containing Chl a (Hofmann et al. 1996). Why the BChl-containing complexes exhibit this long-wavelength feature in their spectra but the complexes containing the other Chl molecules do not, is unclear. Nevertheless, the Soret band and the Qy features of the BChl molecules in the PCP complex are reproduced (Figs. 4E, 5E) assuming only a single prominent feature and a phonon line. Thus, similar to the complexes containing Chl d or 3-acetyl-Chl a, this suggests a highly symmetric environment for the BChls bound in this reconstituted PCP complex.
The Chl–protein interaction induces a blue-shift of the Qy band in the low temperature spectra of Chl a, Chl b and 3-acetyl-Chl a, no shift for Chl d, and a red-shift for BChl a compared to the spectra taken in 2-MTHF low temperature glasses (Figs. 3, 4). This may be explained in a manner similar to that used above to interpret the blue-shifts of the Qy bands induced upon lowering the temperature. Switching from a 2-MTHF glass to the protein may induce a conformational change of the macrocycle that leads to the observed shifts (Gudowska-Nowak et al. 1990). Structural differences between Chl species occur due to different functional groups attached at positions R1 and R2 (Fig. 1), but again, it appears that the R1 site is key. The small aldehyde group of Chl d does not provide sufficient interaction leading to distortion of the macrocycle either in 2-MTHF glass or in the protein. Thus, essentially no protein-induced shift is observed. In contrast, the vinyl group on Ring I of Chl a and Chl b respond to the protein environment and induce a structural change that is manifested spectroscopically as a blue-shift of the Qy band. The red-shift of BChl a in the protein can be attributed to the acetyl group in the position R1. This functional group has been shown to cause a substantial red-shift of absorption spectra of BChl a in LH2 complexes from photosynthetic bacteria (Cogdell et al. 2002). The rotation of the carbonyl group with respect to the plane of Ring I causes changes in π-electron conjugation and this argument has been used to interpret the difference between the B820 and B850 absorbing BChls in different LH2 complexes (Cogdell et al. 2002). However, if this were the sole effect, a protein-induced red-shift would be expected of the 3-acetyl-Chl a that also has an acetyl group in the R1 site. This is not observed. Thus, we must assume that the saturation of the –C=C– bond in Ring II for BChl also plays a role in determining the direction of the shift.
Energy transfer efficiencies
Light absorption by peridinin promotes the molecule from its ground state, S0, to an excited state, denoted S2. The S2 state of peridinin then decays in ∼100 fs to a lower-lying S1 state which cannot be directly accessed from S0 due to symmetry selection rules (Akimoto et al. 1996; Krueger et al. 2001; Zigmantas et al. 2003). Since the lifetime of the S1 state of peridinin in solution was found to be highly dependent on the polarity of the solvent (Bautista et al. 1999; Zigmantas et al. 2001; 2003), an intramolecular charge transfer (ICT) state was invoked to account for this behavior (Bautista et al. 1999), and quantum computations have supported this model (Vaswani et al. 2003). Moreover, it has been argued from pump-dump-probe experiments on peridinin in solution that S1 and ICT states are distinct but connected on a picosecond time-scale (Papagiannakis et al. 2004). However, other time-resolved and two-photon spectroscopic methods have suggested that it is the S1 itself that possesses the charge transfer character (Shima et al. 2003; Zigmantas et al. 2003). Since it is not completely resolved whether the S1 state in PCP is distinct from or the same as the ICT state, we shall use the notation S1/ICT to indicate both possibilities.
The peridinin-to-Chl energy transfer efficiencies for the complexes can be computed assuming a two-state (S2 and S1/ICT) donor model (Fig. 9). The overall efficiency, ε, is given by Desamero et al. 1998:  where kET2 and kET1 are the rate constants for energy transfer from the S2 and S1/ICT states of peridinin, kIC2 is the rate constant for internal conversion from S2 to S1/ICT state and kIC1 is the rate constant for S1/ICT to S0 internal conversion. The rate constants obtained directly from the ultrafast, time-resolved optical spectroscopic experiments carried out (Polívka et al. 2005) are shown in Table 2. The value for the rate constant for S2 to S1/ICT internal conversion, kIC2, is assumed to be 15 ± 1.5 ps−1 (τIC2 = 66 ± 6 fs) from fluorescence up-conversion measurement on the native PCP complex (Linden et al. 2004). The value for kIC1 is assumed to be 0.063 ps−1 (τIC1 = 16 ps) from a measurement of the efficiency and dynamics of energy transfer in the native PCP complex (Zigmantas et al. 2003). The overall energy transfer efficiencies calculated from Eq. 1 are compared with the values obtained here from the steady-state measurements (Fig. 7) and summarized in Table 2. As one can see from the table, the efficiencies determined using both techniques are high in all cases and also in very reasonable agreement with each other.
Fig. 9Schematic diagram of energy levels and energy transfer pathways between peridinin and Chl in the PCP complexes. kET1 and kET2 (solid lines) are rate constants for energy transfer from the S2 and S1/ICT states of peridinin to Chl. kIC2 and kIC1 (dashed lines) are rate constants for internal conversion from S2 to S1/ICT and S1/ICT to S0, respectivelyTable 2Rate constants and energy transfer efficiencies obtained from the ultrafast, time-resolved optical spectroscopic experiments on the PCP complexes reconstituted with various ChlsPCP complexkET2 (ps−1)kIC2 (ps−1)kET1 (ps−1)kIC1 (ps−1)ε (from fluorescence excitation), %ε (from time-resolved absorption), %Chl a10 ± 2.515 ± 1.50.34 ± 0.0340.06394 ± 291 ± 7Chl b10 ± 2.515 ± 1.50.17 ± 0.0170.06392 ± 284 ± 14Chl d13 ± 3.015 ± 1.50.59 ± 0.0590.06396 ± 395 ± 2aChl a11 ± 2.815 ± 1.50.45 ± 0.0450.06399 ± 193 ± 4BChl a10 ± 2.515 ± 1.52.22 ± 0.220.063∼10098 ± 2kET2 and kET1 are the rate constants for energy transfer from the S2 and S1/ICT states of peridinin (Polívka et al 2005). kIC2 and kIC1 are the rate constants for internal conversion from S2 to S1/ICT and from S1/ICT to S0, respectively. kIC2 was obtained from fluorescence up-conversion measurement on the native PCP complex (Linden et al 2004). kIC1 was deduced from the efficiency and dynamics of energy transfer in the native MFPCP given in Ref. (Zigmantas et al 2002). kIC2 and kIC1 were assumed to be unchanged in all the PCP complexes. The peridinin-to-Chl energy transfer efficiency, ε, was obtained from steady-state fluorescence excitation experiments and from ultrafast, time-resolved absorption spectroscopic measurements at room temperature. The uncertainties in the values of ε from fluorescence excitation experiments were derived from the standard deviations obtained in the 450–515 nm wavelength range. The uncertainties in the values of ε from time-resolved absorption measurements were obtained by propagating the published errors in the dynamicsaChl a 3-acetyl-Chl a
In this work, it was shown that optical spectroscopy carried out at low temperatures provides a more highly resolved view of the spectral features of light-harvesting complexes than seen at room temperature. The steady-state optical spectroscopic methods applied to PCP complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either Chl a, Chl b, Chl d, 3-acetyl-Chl a or BChl a illustrate the robust character of the PCP protein which maintains high efficiency of peridinin-to-chlorophyll energy transfer function in the presence of non-native Chl species. The efficiencies of energy transfer deduced from the steady-state measurements are in good agreement with those from ultrafast, time-resolved experiments. The PCP complex continues to be an effective tool for elucidating the molecular details of photosynthetic light-harvesting via systematic modification of its structural components.

reconstitution
chlorophyll
low temperature
absorption
fluorescence
fluorescence excitation
energy transfer efficiency
peridinin-chlorophyll a-protein