Bacterial overgrowth affects urinary proteome analysis: recommendation for centrifugation, temperature, duration, and the use of preservatives during sample collection.
Author(s): Thongboonkerd V, Saetun P
Publication: J Proteome Res, 2007, Vol. 6, Page 4173-81
PubMed ID: 17924682 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of centrifugation and preservation with boric acid or sodium azide on bacterial growth and proteome profile during storage of urine at 4˚C and room temperature.
Conclusion of Paper
In the absence of preservatives, bacterial growth was evident in uncentrifuged urine stored at room temperature and 4˚C after 8 and 20 h, respectively, but bacterial growth was first observed after 12 h at room temperature and was not observed after 48 h of storage at 4˚C when urine was centrifuged to remove debris. Bacterial growth occurred more rapidly when specimens were stored at room temperature than 4˚C, when urine was uncentrifuged than centrifuged, and in unpreserved urine than urine preserved with sodium azide or boric acid. Importantly, addition of 200 mM boric acid completely inhibited bacterial growth during room temperature storage of urine for at least 48 h and use of 10 mM sodium azide delayed any effect of storage on bacterial growth such that it was only evident after 48 h. The number of spots on 2D gels increased as the bacterial growth increased and MALDI-TOF mass spectrometry confirmed that the additional spots were due to bacterial contamination.
Studies
-
Study Purpose
This study investigated the effects of centrifugation and preservation with boric acid or sodium azide on bacterial growth and proteome profile during storage of urine at 4˚C and room temperature. Urine from five healthy patients was pooled and immediately aliquoted. Half of the aliquots were centrifuged at 1500 x g for 15 min to remove cell debris while the other half remained uncentrifuged. Aliquots were left unpreserved or preserved with sodium azide (0.1, 1, or 10 mM) or boric acid (2, 20, or 200 mM) and stored at room temperature or 4˚C for up to 48 h. Bacterial growth was determined by spectrophotometer and confirmed by gram staining. The protein profile of unpreserved urine and urine that was preserved with boric acid was determined by 2D-gel electrophoresis and proteins were identified by MALDI-TOF MS.
Summary of Findings:
In the absence of preservatives, bacterial growth was evident in uncentrifuged urine stored at room temperature and 4˚C after 8 and 20 h, respectively, but bacterial growth was first observed after 12 h at room temperature and was not observed after 48 h of storage at 4˚C when urine was centrifuged to remove debris. Bacterial growth occurred more rapidly when specimens were stored at room temperature than 4˚C, when urines was uncentrifuged than centrifuged, and in unpreserved urine than urine preserved with sodium azide or boric acid. Addition of 200 mM boric acid completely inhibited bacterial growth during room temperature storage of urine for at least 48 h and use of 10 mM sodium azide delayed any effect of storage on bacterial growth such that it was only evident after 48 h.
No changes in the proteome profile were noted after the first 8 h of room temperature storage of centrifuged urine but additional spots appeared with further storage such that by 48 h there were greater than 500 additional spots and 4-fold more total protein (60µg versus 245 µg). Importantly, no new spots appeared when centrifuged urine was preserved with boric acid before room temperature storage. MALDI-TOF MS confirmed that the new spots that appeared during room temperature storage of unpreserved urine were bacterial proteins.
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Spectrophotometry Protein MALDI-TOF MS Protein 1D/2D gels Cell count/volume Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifuged
Not centrifuged
Storage Storage temperature Room temperature
4˚C
Storage Storage duration 0 h
4 h
8 h
12 h
16 h
20 h
24 h
48 h
Biospecimen Preservation Type of fixation/preservation Boric acid
Sodium azide
None (fresh)