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Proteomics and Protein Markers |
1-Microglobulin, and IgG
Departments of1 Clinical Biochemistry and 2 Anaesthesiology, Amager Hospital, Copenhagen, Denmark.
aAddress correspondence to this author at: Department of Clinical Biochemistry, Amager Hospital, Italiensvej 1, DK2300 Copenhagen S, Denmark. Fax 45-3234-3997; e-mail magid{at}dadlnet.dk.
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Abstract |
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1-acid glycoprotein), albumin, 1-microglobulin (protein HC), and IgG to obtain quantitative and temporal data on these 4 proteins. Methods: Urine samples were collected at daily intervals for up to 23 days from 6 patients with surgery-induced inflammation and at hourly intervals for a 24-h period from 7 sepsis patients. Urinary protein concentrations were assessed by immunoturbidimetry.
Results: During surgery-induced inflammation, the increase and decrease in orosomucoid excretion mirrored changes in plasma C-reactive protein. Values for all 4 urinary proteins were increased in sepsis patients. The observed maximum increases in urinary protein excretion relative to the upper reference values were 280-fold for orosomucoid, 98-fold for 1-microglobulin, 33-fold for albumin, and 26-fold for IgG.
Conclusions: Orosomucoid, usually present in plasma and urine in much lower concentrations than albumin, is increased in urine to concentrations equal to or higher than albumin in proteinuria associated with acute inflammation. The pathophysiologic mechanisms responsible for this markedly increased excretion are unknown. Monitoring of urinary excretion of orosomucoid and other specific proteins, expressed as protein/creatinine ratios, may provide a window for clinically relevant real-time observation of changes in acute inflammatory processes. Orosomucoid in urine may be a more informative marker than albumin for inflammation.
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Introduction |
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1-acid glycoprotein)(1)(5)(6). Excess excretion of urinary proteins in the absence of renal disease, so-called functional proteinuria(7), is a common occurrence, but the pathophysiologic mechanisms responsible for this type of proteinuria are not known. Monitoring the urinary excretion of specific proteins in acute disease may provide information of clinical relevance, given future sufficient pathophysiologic insight and availability of appropriate technology. Gosling and coworkers (2)(8) have presented findings indicating that proteinuria occurring 2–4 days after trauma coincides with an increase in plasma C-reactive protein (CRP), and that albuminuria present within 15 min of admission to the intensive care unit (ICU) shows promise as a predictor of outcome. These authors stated that microalbuminuria is a marker of systemic capillary permeability, a view that has been rejected by Molnar and coworkers(9)(10), who argued that microalbuminuria in the critically ill is an acute marker of an inflammatory insult rather than a marker of generalized capillary leakage.
The objectives of the present study were to obtain quantitative data on the intraindividual rates and magnitude of variations in urinary protein excretion in acute disease and to explore the potential usefulness of these data for monitoring of patients. We monitored the excretion in urine of 4 specific proteins: 1-microglobulin, also called protein HC (26.7 kDa), an established marker of renal tubular function (11)(12); albumin (69 kDa) and IgG (150 kDa), established markers of glomerular permeability(7); and orosomucoid (42 kDa). Orosomucoid is an acute-phase reactant assumed to have immunomodulatory and antiinflammatory effects(13) as well as a role in maintaining barriers for transendothelial transport of macromolecules(14).
We describe observations from 10 patients undergoing elective abdominal surgery and 7 patients with sepsis. We used a high-frequency (hourly) sampling strategy to monitor the sepsis patients because we anticipated marked and rapid intraindividual variations based on our earlier observations in patients with acute cardiac disease (15). Our subsequent studies showed that orosomucoid is a prominent component of proteinuria in acute heart disease(16). Our interest in studying the excretion of urinary orosomucoid in states of acute inflammation was further strengthened by our recent finding that the urinary orosomucoid excretion rate is a strong risk marker for cardiovascular mortality in patients with type 2 diabetes and appears to be a marker of low-grade inflammation in this chronic condition(17)(18).
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Materials and Methods |
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The first group, sepsis patients (n = 7) representing cases of severe inflammation, were monitored for a period of 24 h. To identify possible short-term variations, we collected urine samples for analysis at hourly intervals from a urinary bladder catheter inserted as a routine clinical intervention.
The second group of patients (n = 6), representing cases with an expected inflammatory response to surgery (10), were studied from the day before the surgical intervention for up to 23 days after surgery to monitor changes in urinary protein excretion from the beginning to the end of an inflammatory event. Data from catheter urine samples obtained within the first 4 h after surgery were used for comparison with results from catheter urine obtained from the sepsis patients. Subsequently, spot urine samples obtained either from a catheter or by spontaneous micturition were used for analysis.
In both patient groups, routine blood analyses, including albumin, orosomucoid, and CRP, were performed to assess the inflammatory state of the patient.
patients
In the period from August 2003 to October 2004, a total of 17 patients at Amager Hospital (Copenhagen) were selected for inclusion in this study. Ten patients were enlisted before planned abdominal surgery for colon cancer (n = 8) or colon diverticulitis (n = 2). Seven patients were under treatment for sepsis in the ICU; their diagnoses and classification were based on generally accepted criteria (19)(20). Medical histories were obtained retrospectively from the medical records. None of the patients included had a diagnosis of urinary tract infection. Patient characteristics are summarized in Table 1
, and brief excerpts from some of the case records are given below.
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methods
Handling of specimens.
Urine samples were obtained as midstream urine by spontaneous micturition or from a closed diuresis measuring system (UnoMeter 500TM; Unomedical A/S) attached to a urinary bladder catheter. In the latter case, urine was allowed to remain for periods of 
1 h in the bladder; the urine produced was then transferred to the collection bag, and a representative sample (5 mL) was taken from the transfer tube. The time of transfer and the urine volume (milliliters) was recorded by the attending nurse according to written instructions and used for the calculations described below. Urine samples were kept at ambient temperature at the wards and brought to the laboratory within 10 h after sampling, where they were kept refrigerated (2–8 °C) without additives for <10 days before analysis. Blood samples were taken by venipuncture or from a central venous catheter, brought to the laboratory, and analyzed within 10 h of sampling.
Urine analyses.
Orosomucoid was analyzed with a newly developed, high-sensitivity particle-enhanced turbidimetric assay (21). Albumin, 1-microglobulin, and IgG were measured by immunoturbidimetry (Integra 700/800 or Cobas Mira; Roche) using monospecific rabbit antisera from DakoCytomation for the 2 latter proteins. The detection limits (intraseries imprecision, as CVs) for these proteins in urine were as follows: orosomucoid, 0.05 mg/L (1% at 1.08 mg/L); albumin, 3.3 mg/L (4.5% at 11 mg/L); 1-microglobulin, 5 mg/L (4.9% at 12 mg/L); and IgG, 7 mg/L (4.1% at 47.7 mg/L). Total protein was determined by colorimetry using the biuret reaction and creatinine by an enzymatic method using creatininase, both from Roche.
Blood analyses.
Orosomucoid and albumin were measured by immunoturbidimetry; CRP was measured by a particle-enhanced turbidimetric immunoassay.
All protein analyses in urine and blood where calibrated with Human Serum Protein Calibrator (code no. X 0908; DakoCytomation), traceable to the International Certified Reference Material (CRM 470), with the exception of 1-microglobulin, which was calibrated by use of DakoCytomation calibrator X-0938.
statistics
Statistical analyses were performed with Statistica for Windows, Ver. 6 (StatSoft Inc.). A Mann–Whitney U-test was used for the comparison of nongaussian-distributed variables between the 2 groups. The protein excretion rate (in µg/min) was calculated as the protein concentration (mg/L) times the volume (mL) divided by the duration of the collection period (min). Protein excretion, in mg/24 h, for each of the sepsis cases was calculated from the median protein excretion rate as µg/min x 1440 min.
The protein/creatinine ratio was calculated from the concentration of the protein (mg/L) and the concentration of creatinine (mmol/L) as mg protein/mmol creatinine. Values for the protein/creatinine ratio given as mg/mmol can be converted to mg/g by multiplication by 8.84. The microalbuminuric range, 2.5–25 mg/mmol, is also defined as 30–300 mg/g, 30–300 mg/24 h, and 20–200 µg/min (22).
ethics
The local ethics committee in Copenhagen approved the study protocol (KF 01-023/03), and the study was carried out in accordance with Helsinki Declaration II.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The protein/creatinine ratios for all 4 proteins examined in urine were increased in both groups. In the sepsis group, the relative increase in protein/creatinine ratio (range of maximum values) was 41- to 280-fold for orosomucoid, 8- to 98-fold for 1-microglobulin, 3- to 33-fold for albumin, and 3- to 26-fold for IgG. The maximum values for the albumin/creatinine ratio were above the microalbuminuric range in 4 cases and within that range in 3 cases. The orosomucoid and albumin values obtained from the surgery group 1 to 4 h after surgery were significantly lower than those in the sepsis group but were increased compared with the reference values. Total protein excretion remained <1.2 g/L in all cases (data not shown).
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In plasma, the CRP and orosomucoid concentrations were significantly higher, and that of albumin lower, in the sepsis group than in the surgical intervention group.
Calculated values for protein excretion in mg/24 h for the 7 sepsis cases were within the following ranges: orosomucoid, 22–255; 1-microglobulin, 40–94; albumin, 19–88; and IgG, <7 to 44.
monitoring of patients with surgery-induced inflammation
The results from monitoring of 6 patients for at least 6 days after surgery showed peak values for the albumin/creatinine ratio, reached between days 2 and 5 after surgery, of 5.6–22.9 mg/mmol, which is within the microalbuminuric range. The corresponding values for orosomucoid/creatinine ratio were 1.3–21.2 mg/mmol. In all of the cases, orosomucoid excretion in urine increased and decreased together with CRP in plasma. On days 6 to 8 after surgery, plasma CRP returned to concentrations <35 mg/L, and orosomucoid/creatinine ratios to values <2 in 3 cases. In these cases, exemplified by case A in Fig. 1
, the postoperative period was clinically uneventful and the patients were discharged on schedule.
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In case B (Fig. 1
), a minor cerebral thrombosis occurred at day 2, on day 4 antibiotic treatment was initiated, and on day 5 a tracheotomy was performed to allow drainage of bronchial secretion. The tracheotomy was removed on day 12; subsequently, the patient became afebrile and recovered. In this case, orosomucoid and albumin excretion rates were of the same order of magnitude, and both covaried with the changes in the CRP concentration in plasma.
monitoring of sepsis patients
Results from 3 sepsis cases obtained by monitoring of urinary protein excretion hourly over a 24-h period are shown in Fig. 2
. The relative increases in excretion differed for the 4 proteins and were particularly marked for orosomucoid. The magnitudes of these differences are emphasized in Fig. 2
by the reporting of protein/creatinine ratios as multiples of the corresponding upper reference values obtained from healthy individuals.
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Case C suffered from peritonitis caused by a perforated gastric ulcer. In this case, the protein/creatinine ratio for orosomucoid was 4-fold higher than that for albumin.
During the monitoring periods, orosomucoid excretion decreased in case D and increased in case E. Case D, who suffered from diabetic nephropathy and arterial hypertension, was recovering from sepsis under antibiotic treatment; the patient was afebrile and clinically in fair condition the day after the monitoring period. In case E, the patient died the day after the monitoring period from peritonitis and septic shock.
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Discussion |
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temporal relationship to inflammatory activity
CRP in plasma is currently recognized and widely used as the preferred marker of inflammatory activity. The findings exemplified in Fig. 1
indicate that the urinary excretion of orosomucoid increases and decreases in a temporal relationship to CRP in plasma. We did not perform simultaneous sampling of plasma and urine and therefore are unable to determine whether increases in plasma CRP and urinary orosomucoid coincide or whether one of these components precedes the other in time. The observations suggest to us that monitoring of urinary excretion of orosomucoid, and possibly other specific proteins, may be as informative as plasma CRP with regard to assessment of acute inflammatory activity.
description and classification
The observed proteinuria can be characterized as small (<1.2 g/L) and reversible, as found in the group of surgery patients, and mixed glomerular-tubular, comprising both the established markers of glomerular function (albumin and IgG) as well as a marker of proximal tubular function (1-microglobulin). In general, the proteinuria of nonrenal, acute disease states (1)(2)(3)(4)(23)(23)(24)(25)(26) as well as postexercise proteinuria(5)(27) may be of a mixed tubular-glomerular type, but systematic studies using a standardized set of markers are required to allow firm conclusions. Our finding in sepsis patients of large increases in the excretion of the acute-phase reactant orosomucoid has not, to the best of our knowledge, been described previously. In healthy individuals, orosomucoid is present in urine in amounts 10- to 20-fold lower than albumin, but in our sepsis patients, orosomucoid excretion reached rates equal to or higher than those of albumin. The observed increase in urinary albumin excretion to amounts within the microalbuminuric range are in quantitative agreement with previous reports on sepsis patients(8) and on patients undergoing abdominal surgery(9).
We were particularly interested in seeing whether proteinuria during inflammation was continuous, as is commonly assumed, or intermittent (episodic), a possibility raised by unconfirmed observations in patients with acute cardiac diseases (15)(16). As we observed in case D (Fig. 2
), changes in albumin excretion may indeed vary within a few hours between near-normal and 10-fold–increased values. Evidently, the use of 24-h collection periods, a former gold standard for quantifying protein excretion, would hinder the observation of changes that may occur within that period of time. The frequency of urine sampling needed for effective monitoring must be determined by the possible clinical significance that may be ascribed to observations of rapid changes.
We calculated and plotted protein excretion in the sepsis cases as excretion rates in milligrams per minute (data not shown) and found higher variability than for results reported as protein/creatinine ratios. This, we presume, is largely attributable to the added uncertainty associated with collection times and urine volume.
We conclude that a simpler sampling procedure based on spot urines obtained several times per day rather than hourly, timed urine samples is likely to be sufficiently informative for the purpose of monitoring urinary protein excretion in sepsis patients.
pathophysiologic mechanisms
In 1986, Gosling and Sutcliffe (2) raised the question as to why trauma, sometimes at a site distant from the kidney, can produce immediate glomerular-tubular proteinuria. Presumably, protein handling by the nephron may be influenced by substances generated by the inflammatory cascade, the common denominator of an array of clinical conditions associated with functional proteinuria. One may ask whether such substances can now be identified and whether their actions affect glomerular permselectivity or tubular reabsorption of proteins. Likely candidates include interleukin-6 and other cytokines that have been associated with proteinuria in acute as well as in chronic diseases(28)(29)(30). Pedersen and Sorenson(30) found a direct correlation between microalbuminuria and proinflammatory cytokines in malignancies, indicating a pathogenic relationship between inflammation and glomerular leakage of albumin. Interleukin-6 has been labeled "the exercise factor" and has been shown to increase up to 100-fold in blood during exercise(31). In experimental studies, proteinuric effects of tumor necrosis factor and natriuretic peptides have been demonstrated(32)(33).
A common mechanism may be responsible for the observed increase in urinary excretion of all 4 of the proteins studied. The glomerular membrane and the proximal tubuli both appear to be involved. Protein transport at these sites is incompletely understood and much debated (34)(35)(36), and further research is needed to identify the causative factors of proteinuria associated with inflammation.
clinical aspects: acute and chronic inflammatory conditions
In their prospective observational study, Gosling et al. (8) compared the predictive value of low-level urinary albumin excretion with acute physiology scores and concluded that microalbuminuria shows promise as a predictor of outcome for surgical, trauma, and burn patients in the ICU. The present observations indicate that the relative increase in urinary orosomucoid excretion during acute inflammation is much higher than that of albumin. In spite of the finding that a high degree of covariation between the urinary concentrations of these 2 proteins may occur, more reliable information may be provided by measuring orosomucoid because of the amplification of the signal and a favorable signal-to-noise ratio. As a consequence, we propose that orosomucoid should be included in future studies on the clinical utility of urinary protein excretion in patients with acute inflammatory conditions.
In a recent review on resolving the diagnostic challenges of sepsis, Carrigan et al. (37) concluded that a technology for real-time immune status monitoring is needed. Our results indicate that rapid changes in immune status are reflected by changes in urinary protein excretion, expressed as protein/creatinine ratios. Urine may be considered as a possible supplement or alternative to blood as sampling material in future studies of monitoring systems to be developed for this purpose.
In chronic disease, urinary albumin is used as an established marker of cardiovascular risk. As summarized by Paisley et al. (38), proteinuria is associated with vascular risk and a systemic increase in vascular permeability. Endothelial dysfunction occurs early in atherosclerosis and modulates vascular permeability. Vascular risk and chronic inflammation are associated. In patients with type 2 diabetes, albuminuria is associated with signs of low-grade chronic inflammation(39). We have recently demonstrated that orosomucoid in urine is a powerful predictor of cardiovascular mortality even in normoalbuminuric patients with type 2 diabetes(18). These results, together with the findings presented above, indicate that urinary orosomucoid may be a more informative marker than urinary albumin of acute as well as chronic inflammation.
The present study included a small number of cases, but we believe that our observations of a consistent and marked increase in urinary orosomucoid excretion during acute inflammation and the covariation of CRP in plasma with the urinary excretion of orosomucoid and other proteins are noteworthy.
In conclusion, the observations presented above indicate that measurements of urinary excretion of orosomucoid, albumin, and other proteins, expressed as protein/creatinine ratios, may reflect even rapid changes in the inflammatory cascade. Further research is needed to clarify the pathophysiologic mechanisms involved. It appears worthwhile to investigate whether urinary orosomucoid is a more informative marker of inflammation than urinary albumin and whether measurements of orosomucoid and other proteins in urine can add information of clinical value to the established markers of inflammation, such as CRP in plasma.
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Acknowledgments |
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References |
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increases albumin permeability of isolated rat glomeruli through the generation of superoxide. J Am Soc Nephrol 1998;9:433-438.[Abstract]
1-antitrypsin. Scand J Clin Lab Invest 1996;56:691-700.[Web of Science][Medline]
[Order article via Infotrieve]The following articles in journals at HighWire Press have cited this article:
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  D. Sviridov, B. Meilinger, S. K. Drake, G. T. Hoehn, and G. L. Hortin Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin Clin. Chem., March 1, 2006; 52(3): 389 - 397. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, orosomucoid; 
, albumin; 
, CRP. See text for excerpts of case records.
, IgG. See text for excerpts of case reports.