Differential protein expression in serum of abdominal aortic aneurysm patients - a proteomic approach.

OBJECTIVE: The aim of the study is to investigate the differential expression of proteins in serum of abdominal aortic aneurysm (AAA) patients in relation to aneurysm size (D(max)) and progression. METHODS: Two-dimensional differential in-gel electrophoresis (2D-DIGE) together with tandem mass spectrometry (MS/MS) was used to analyse the serum proteome from patients with small (D(max) 30-54 mm) AAA, either stable (increase D(max) <5 mm year⁻¹; n = 8) or progressive (increase D(max) ≥5 mm year⁻¹; n = 8), and large (D(max) ≥ 55 mm; n = 8) AAA. The identified proteins were quantitatively validated in a larger population (n = 80). RESULTS: Several proteins were differentially expressed in serum of small stable, small progressive and large AAA. Three validated proteins (immunoglobulin G (IgG), α1-antitrypsin (α1-AT) and Factor XII activity) showed strong correlation with D(max). Size combined with either Factor XII activity or α1-antitrypsin had minimal effect on the prognostic value in predicting aneurysm progression compared with size alone (area under the curve (AUC), 0.85; 95% confidence interval (CI), 0.73-0.97; p < 0.001 and AUC, 0.85; 95% CI, 0.72-0.98; p < 0.001 vs. AUC, 0.83; 95% CI, 0.71-0.96; p < 0.001, respectively). CONCLUSION: The present study indicates that both Factor XII and α1-antitrypsin are found in increased amounts in the serum of patients with expanding AAA. However, combination of either Factor XII or α1-antitrypsin with aneurysm diameter had little effect on prediction of aneurysm progression versus diameter alone.

Three-dimensional flow-through protein platform.

We have developed a new protein microarray (ImmunoFlow Protein Platform, IFPP) that utilizes a porous nitrocellulose (NC) membrane with printed spots of capture probes. The sample is pumped actively through the NC membrane, to enhance binding efficiency and introduce stringency. Compared to protein microarrays assayed with the conventional incubation-shaking method the rate of binding is enhanced on the IFPP by at least a factor of 10, so that the total assay time can be reduced drastically without compromising sensitivity. Similarly, the sensitivity can be improved. We demonstrate the detection of 1 pM of C-reactive protein (CRP) in 70 microL of plasma within a total assay time of 7 min. The small sample and reagent volumes, combined with the speed of the assay, make our IFPP also well-suited for a point-of-care/near-patient setting. The potential clinical application of the IFPP is demonstrated by validating CRP detection both in human plasma and serum samples against standard clinical laboratory methods.

Release kinetics of intact and degraded troponin I and T after irreversible cell damage.

PURPOSE: We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage. METHODS: Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0-12 h) and irreversible phase (12-30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products. RESULTS: During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used. CONCLUSIONS: Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.

[Real change or natural fluctuation?] / Echte verandering of natuurlijke schommeling?

When monitoring patients over time, it may be difficult to distinguish 'real changes' from so-called 'natural fluctuations' when interpreting consecutive laboratory results. Consider a patient whose cholesterol level has decreased from a baseline 6.6 mmol/L to 6.1 mmol/L six months after receiving lifestyle advice. How likely is it that this is a 'real change', reflecting a lifestyle change, rather than random fluctuation? Physicians mostly rely on their intuition and clinical experience when interpreting changes in consecutive laboratory results. For inexperienced physicians, the lack of an easy reference for the interpretation of consecutive laboratory results can make decision-making challenging. We have developed the medical/educational smartphone app Labtracker+ that calculates the probability of a 'real change' between two consecutive laboratory results, using biological variation data from scientific literature and analytical precision that is achieved in contemporary laboratories. This approach may complement intuitive, experience-based interpretations of consecutive laboratory results.

C-reactive protein, atherosclerosis and kidney function in hypertensive patients.

Previous studies have shown a relationship between coronary or carotid atherosclerosis and C-reactive protein (CRP) concentrations. In the present investigation, we evaluated the relationship between high-sensitivity CRP (hsCRP) concentrations and the presence of atherosclerotic lesions in the renal arteries and/or abdominal aorta. In 95 hypertensive patients who underwent intra-arterial DSA on suspicion of renovascular disease, blood was sampled during the procedure for measurement of hsCRP. The presence of atherosclerotic lesions was assessed at the level of the renal arteries and the abdominal aorta. Haemodynamically significant renal artery stenosis was diagnosed when 50% or more stenosis was observed. Patients with fibromuscular disease (n = 8) or incomplete data (n = 4) were excluded from analysis. The results revealed that the median hsCRP concentrations were significantly higher among the 57 patients with atherosclerosis of the aorta and/or renal arteries compared to those in the 26 patients without any angiographic lesions (4.6 vs 1.7 mg/l; P < 0.005). Moreover, in patients with renal artery stenosis, levels of hsCRP were higher when the degree of stenosis exceeded 50%. However, the association between hsCRP and the presence of atherosclerosis appeared to be confounded by serum creatinine, creatinine clearance, age and gender. In the whole group a significant inverse relationship was found between creatinine clearance and hsCRP (P < 0.05). In conclusion, hsCRP concentrations are related to atherosclerotic lesions in the renal arteries and the abdominal aorta. While this supports the view that atherosclerotic renal artery stenosis is part of a systemic inflammatory vascular disease, increased concentrations of CRP may also coincide with decreased renal function.

Enhanced preoperative C-reactive protein plasma levels as a risk factor for postoperative infections after cardiac surgery.

BACKGROUND: We examined the possible predictive role of preoperative C-reactive protein (CRP) levels for postoperative infections in patients who have cardiac operations. METHODS: CRP levels were determined on the day before the operation and on postoperative days 1 to 4 and 6 in 593 consecutive patients. Furthermore, we documented infectious disease-related data. RESULTS: Patients in whom an infection developed during the postoperative course (n = 87) had significantly higher CRP levels on the day before operation (17.8+/-3.9 mg/L compared with 7.7+/-0.7 mg/L; p<0.001) and on postoperative days 4 and 6. The incidence of postoperative infections was significantly higher in patients with increased preoperative CRP levels than in those with normal preoperative CRP levels (25.3% versus 11.2%, respectively; p<0.001). Furthermore, patients with higher preoperative CRP levels had a significantly longer postoperative hospital stay than those with normal preoperative CRP levels (10.8+/-1.2 days versus 7.8+/-0.3 days; p<0.001). Multivariate analysis, including classic risk factors and increased preoperative CRP levels, demonstrated that higher preoperative CRP was the most important variable predicting postoperative infection (odds ratio = 2.7; 95% confidence interval = 1.7 to 4.3; p<0.001). CONCLUSIONS: Patients with higher preoperative CRP levels are at increased risk for postoperative infections. Therefore, preoperative measurement of CRP might be a useful, predictive marker in risk stratification for postoperative infections in patients scheduled for cardiac operations.

Usefulness of monitoring beta-glucuronidase in pleural effusions.

BACKGROUND: The objective of the study was to evaluate the additional value of beta-glucuronidase (BGD), a lysosomal enzyme in the analysis of transudative and exsudative pleural effusions, especially between malignant and non-malignant effusions. DESIGN AND METHODS: Pleural fluid samples obtained from four respective diagnostic groups: transudates parapneumonic effusions, malignant effusions or pleuritis carcinomatosa, and empyema were evaluated. RESULTS: Beta-glucuronidase was significantly different between transudative and exsudative effusions (p<0.001) as well as between parapneumonic and malignant effusions (p<0.03), parapneumonic effusions and empyema (p<0.002), and malignant and empyema (p<0.002), respectively. Logistic regression analysis yielded a weak discrimination between the parapneumonic and malignant groups. CONCLUSIONS: Beta-glucuronidase activity differed between pleural effusions of various origin. However, including BGD in the biochemical work-up of pleural effusions did not reveal discriminatory value in the assessment of the classification of these effusions.

Serum beta-glucuronidase activity in a population of ex-coalminers.

BACKGROUND: The aim of this study was to investigate whether BGD activity is of additional value in the assessment of pulmonary inflammation caused by coal dust exposure. DESIGN AND METHODS: Ex-coalminers were included in this study. Forty-eight healthy male subjects, without a relevant medical history, were used as controls. RESULTS: In ex-coalminers serum BGD activity was higher compared to the control group. Moreover, ex-coalminers with a normal chest radiograph and normal serum LDH demonstrated elevated serum BGD compared to the control group. However, no relation was found in the total group of ex-coalminers between serum BGD activity and pulmonary function parameters. CONCLUSIONS: Our study adds in vivo human evidence to the already existing animal data that BGD is a potential biomarker useful in monitoring pulmonary inflammation caused by coal dust exposure.

A comparative study on the diagnostic value of prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) in patients with carcinoma of the prostate gland.

Serum prostatic-specific antigen (PSA) and prostatic acid phosphatase (PAP) were determined simultaneously in 241 patients presented to the Urology Department. The patients consisted of 140 prostatic carcinoma patients (34 newly diagnosed and 106 previously treated) and 101 patients with benign prostatic hypertrophy (BPH). Prostatic acid phosphatase was measured by two different methods, an enzymatic method (PAP-EA, Boehringer) with tartrate inhibition and an immunoenzymetric assay (PAP-IEMA, Hybritech). The concentration of prostatic specific antigen in serum was measured using a recently introduced immunoradiometric assay (PSA-IRMA, Hybritech). Receiver operating characteristic curves were constructed to compare the diagnostic value of the different tests at different cutoff values. The diagnostic efficiencies of the PAP-EA and the PAP-IEMA appeared to be similar. A better diagnostic efficiency for PSA compared to PAP was found independent of the cutoff value. The upper-normal limit of 2.7 micrograms/l for PSA, as suggested by the manufacturer and mentioned in the literature introduces too many false-positive results. We therefore selected 10 micrograms/l as the upper-normal limit for PSA (sensitivity 57%, specificity 88%). Combined sensitivity found for PAP + PSA was 37% with a specificity of 97%. A literature survey is included to allow better comparison with data published elsewhere.

The diagnostic value of urinary transferrin compared to serum prostatic specific antigen (PSA) and prostatic acid phosphatase (PAP) in patients with prostatic cancer.

Urinary transferrin, serum prostatic acid phosphatase (PAP) and prostatic-specific antigen (PSA) concentrations were measured in patients with prostatic cancer, prostatitis, benign prostatic hypertrophy (BPH) and in a control group. In contrast to recently published data it is concluded that urinary transferrin is not suitable as a tumor marker for prostatic carcinoma. Receiver Operating Characteristic curves were constructed to compare the diagnostic value of the different tests at different cutoff values. Sensitivity and specificity of the urinary marker are extremely low compared to the serum markers PAP and especially PSA, making the former not suitable as an additional marker.

Thrombolytic therapy does not change the release ratios of enzymatic and non-enzymatic myocardial marker proteins.

Measurements of cardiac marker proteins in plasma from patients with acute myocardial infarction (AMI) have become important in the evaluation of recanalization therapy. The validity of this approach has however been questioned, because it was claimed that coronary reperfusion may increase the recovery in plasma of cardiac enzymes, such as creatine kinase (CK). In the present study, possible effects of thrombolytic therapy on the release of enzymatic and nonenzymatic marker proteins were investigated. Activities of CK and lactate dehydrogenase (LDH), and concentrations of myoglobin (Mb) and fatty acid-binding protein (FABP) were determined in serial plasma samples obtained from 50 patients with confirmed AMI, of whom 36 received thrombolytic therapy, and 14 did not. Treatment delay was 2.8+/-1.6 (mean+/-SD) h, and hospital delay in untreated patients was 2.7+/-1.8 h. Average infarct size, expressed in gram-equivalents of heart muscle per litre of plasma (g-eq/l), varied between 5.5 and 7.2 g-eq/l for the four marker proteins in patients treated with thrombolytic therapy, and between 4.6 and 6.4 g-eq/l in untreated patients, with a tendency to larger infarct sizes for Mb and FABP than for CK and LDH. Thrombolytic therapy, although significantly accelerating protein release rates, did not influence the release ratios. These results indicate that thrombolytic therapy has no significant effects on the recovery of cardiac marker proteins in plasma.

Fatty acid-binding protein and the early detection of acute myocardial infarction.

Fatty acid-binding protein (FABP) is a newly introduced plasma marker of acute myocardial infarction (AMI). The plasma kinetics of FABP (15 kD) closely resemble those of myoglobin (18 kD) in that elevated plasma concentrations are found within 3 h after AMI and return to normal generally within 12 to 24 h. This makes both myoglobin and FABP useful biochemical markers for the early assessment or exclusion of AMI. The myocardial tissue content of FABP (0.5 mg/g) is about five-fold lower than that of myoglobin (2.5 mg/g), but the reference plasma concentration of FABP (ca. 2 microg/l) is about 15-fold lower than that of myoglobin (ca. 32 microg/l), together suggesting a superior performance of FABP for the early detection of AMI. Indeed, in a study including blood samples from 83 patients with confirmed AMI, taken immediately upon admission to the hospital (< 6 h after AMI), the diagnostic sensitivity was significantly greater for FABP (78%, confidence interval 67-87%) than for myoglobin (53%, CI 40-64%) (P < 0.05). In addition, the differences in contents of myoglobin and FABP in heart and skeletal muscles and their simultaneous release upon muscle injury allow the plasma ratio of myoglobin/FABP to be applied for discrimination of myocardial (ratio 4-5) from skeletal muscle injury (ratio 20-70). Rapid and sensitive immunochemical assay systems for FABP in plasma are now being developed and soon will enable the introduction of this marker in clinical practice.

Serum lactate dehydrogenase and its isoenzyme pattern in ex-coalminers.

Serum lactate dehydrogenase (LDH) activity, a marker of cell damage, is increased in several pulmonary disorders, especially when fibrosis is involved. In rats exposed to silica, high levels of LDH activity were found. A rise of serum LDH3 has been associated with lung tissue injury. The aim of this study was to investigate the serum LDH isoenzyme pattern after coal-dust exposure and the possible relation to pulmonary function tests. Ex-coalminers (n = 201), with a history of coal-dust exposure more than 20 yr ago, were admitted to the authors' hospital for a medical check-up and were included in the study. The serum LDH activity was found to be elevated in 79.1% of the ex-coalminers (634 +/- 245 U I-1). Moreover, in 196 of the 201 cases (97.5%), a high LDH3 level (31 +/- 4%) was demonstrated. A moderate negative relation was found between the forced expiratory volume in 1 s (FEV1) and the LDH activity (r = -0.26; P < 0.001), as well as between FEV1 and LDH3 activity (r = -0.23; P < 0.001), even in the subgroup (n = 42) with a normal LDH. All other liver function tests were within normal limits. These results suggest that coal-dust, even many years after the actual exposure, causes an increase in the total serum LDH activity and changes in the LDH-isoenzyme pattern, mainly characterized by a high LDH3 activity.

The value of HbA1 and fructosamine in predicting impaired glucose tolerance--an alternative to OGTT to detect diabetes mellitus or gestational diabetes.

Glycosylated haemoglobin and glycosylated serum protein concentrations (fructosamine) have been monitored in patients suspected of diabetes mellitus (n = 183), in pregnant women suspected of gestational diabetes (n = 250) and in control groups (n = 184). The response to the standard 75 g oral glucose tolerance test was used to confirm or reject the diagnosis, using different criteria for detection of diabetes mellitus compared to detection of gestational diabetes. A slightly higher sensitivity was observed for fructosamine compared to glycosylated haemoglobin to detect impaired glucose tolerance (52 vs 44%) or gestational diabetes (17% vs 8%). For detection of diabetic oral glucose tolerance no difference was observed between glycosylated haemoglobin and fructosamine; sensitivity for both parameters was 67%. The results suggest that fructosamine is slightly more sensitive in detecting borderline-abnormal glucose tolerance, whereas no differences are observed for detection of clearly abnormal oral glucose tolerance tests.

Cardiac troponin T release into plasma after acute myocardial infarction: only fractional recovery compared with enzymes.

After acute myocardial infarction (AMI) cardiac enzymes and proteins are released into plasma and are used as biochemical markers of cardiac muscle injury. We studied the completeness of the release of troponin T, a cardiac protein that is largely bound to myofibrillar structures and compared it with the release of cytoplasmic cardiac enzymes in 22 patients with AMI, who were treated with thrombolytic therapy. Creatine kinase (CK; EC 2.7.3.2), hydroxybutyrate dehydrogenase (HBDH), lactate dehydrogenase (LDH; EC 1.1.1.27) and troponin T were assayed serially in plasma samples obtained frequently and for at least 168 h after the start of thrombolytic therapy. Cumulative release of enzymes and troponin T in plasma were calculated by using a two-compartment model for circulating proteins. In order to express the cumulative plasma releases in gram equivalent (g-eq) healthy myocardium per litre plasma (infarct size), we determined HBDH, LDH and total troponin T contents per gram net weight of tissue in 17 human hearts obtained post-mortem from patients who died from non-cardiac causes. Mean (SD) tissue contents per gram wet weight of, respectively, 156 +/- 25 U/g, 385 +/- 59 U/g and 234 +/- 65 micrograms/g were found. For the cardiac enzymes CK, HBDH and LDH the mean (SEM, n = 22) total release over 72 h, was, respectively, 5.9 +/- 1.5, 5.9 +/- 1.6 and 6.1 +/- 1.7 g-eq/L. There was no further increase after 72 h and the differences between enzymes were not significant. The mean (SEM) cumulative troponin T release, expressed in gram equivalents of myocardium per litre of plasma was only 0.30 +/- 0.09 g-eq/L after 72 h and 0.51 +/- 0.61 g-eq/L after 168 h. After 72 h total recovery of troponin T in g-eq/L was only 5% and after 168 h only 8.5% of the total recovery of cytoplasmic cardiac enzymes after 72 h. Cumulative troponin T release after 72 h and after 168 h correlates well with infarct size, estimated from cumulative cytoplasmic enzyme release. However, quantification of infarct size should preferably be performed from plasma release curves of cytoplasmic cardiac enzymes or proteins in order to prevent underestimation of infarct size, caused by incomplete release of the non-cytoplasmic proteins.

Multicentre study of a new test for TSH-screening in blood spots.

A multicentre study for the detection of congenital hypothyroidism by enzyme-immunoassay of thyrotropin in filter paper blood spots is described. The method is accurate, with good precision and adequate sensitivity. No interference due to common blood constituents has been observed. Moreover, the TSH determination can easily be performed by photometric measurement of enzyme activity. Large series of samples can be processed by automation using common laboratory analysers. Values show good agreement with those obtained by RIA methods. We present a method for screening for congenital hypothyroidism which can be performed in all clinical laboratories.

Glycosylated serum proteins and glycosylated haemoglobin in normal pregnancy.

Glycosylated haemoglobin (HbA1) and glycosylated serum protein concentrations (fructosamine) were monitored in 162 normal pregnancies. The response to the standard 75 g oral glucose tolerance test (OGTT) was used to exclude gestational diabetes. A significant increase in glucose intolerance, measured from the area under the OGTT-curve (r = 0.29, P less than 0.0005) and a highly significant decrease in serum albumin concentration (r = -0.79, P less than 0.0005) due to the redistribution of body water, as occurs during pregnancy, resulted in a statistically significant decrease in serum fructosamine concentration (r = -0.23, P less than 0.01) when related to gestational age in weeks.

Is prostate-specific antigen the most useful marker for screening in prostate cancer?

Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) levels were determined in 241 patients attending the Department of Urology. The population consisted of 140 prostate cancer patients (34 newly diagnosed and 106 under treatment) and 101 patients with benign prostatic hypertrophy (BPH). The diagnostic values of PAP measured by enzymatic assay (EA) and by immunoenzymetric assay (IEMA) appeared to be similar. Elevated PAP (IEMA) levels were found in 10% of the patients with BPH and in 38% of the cancer patients. PSA was measured by immunoradiometric assay (IRMA) and receiver operating characteristic curves were constructed to compare the diagnostic benefits of different cutoff values. PSA (10 micrograms/L) reached a specificity of 88% and a sensitivity of 46%. With a cutoff value of 2.7 micrograms/L, the sensitivity increased to 64%, whereas the specificity fell to 58%. It is concluded that PSA is the most useful marker as a screening test.

Serum LDH: a marker of disease activity and its response to therapy in idiopathic pulmonary fibrosis.

In the following case report the determination of serum lactate dehydrogenase (LDH)-a simple though non-specific test-reflects changes of disease activity and clinical improvement during treatment with cyclophosphamide and a tapered dose of prednisone from 100 to 0 mg daily in a 43-year-old woman with idiopathic pulmonary fibrosis. The trend observed in this case indicates that the serum LDH-activity may be directly proportional to the extent of this diffuse inflammatory pulmonary disease.

[Evaluation of clinical-pathological conferences using a program for computer-assisted diagnosis; analysis of 100 case reports]. / Evaluatie van klinisch-pathologische conferenties met behulp van een programma voor 'computer-ondersteunde' diagnose; analyse van een honderdtal ziektegeschiedenissen.

Case studies as weekly published in the New England Journal of Medicine are analysed using the computer programme Medwise. The programme covers more than 3,500 disease profiles. After entering clinical data on all patients described in 1986 and 1987, the diagnostic outcome was compared with the results of the clinicians and with the eventual patho-anatomical findings. Medwise gave the correct diagnosis in 93% of the 104 cases considered. In 15 cases the clinicians failed to formulate the patho-anatomical diagnosis; Medwise correctly detected nine of these (60%). Medwise missed the diagnosis in seven cases, but the clinicians' answers were also faulty in six of them. These results indicate the usefulness of computer-assisted interpretation of clinical findings for establishing the diagnosis, provided that the course and laboratory data concerning the patient are accurately documented.