Prevalence of Different Haptoglobin Phenotypes in Patients With COPD- Frequent Exacerbators Versus Non Exacerbators
Condition: Pulmonary Disease, Chronic Obstructive
MeSH terms: Pulmonary Disease, Chronic Obstructive
Study type: Observational

Summary: 
      Chronic obstructive pulmonary disease (COPD) is a common disease in smokers. COPD has a
      slowly deteriorating course, punctuated by exacerbations- acute events characterized by
      increasing shortness of breath and putrid sputum. Exacerbations of COPD may be precipitated
      by several factors, most commonly infections.

      Exacerbation frequency generally increases with declining lung function. However, some
      patients with COPD consistently experience a higher rate of exacerbations than others despite
      similar severity of COPD. This has led researchers to postulate the existence of a distinct
      subgroup of "frequent exacerbators" . Recent work has also brought attention to a subset of
      patients who experience remarkably few exacerbations despite significantly impaired lung
      function. Careful characterization of both of these extreme subgroups of COPD may offer
      additional insights into why certain patients are prone to frequent exacerbations while
      others remain relatively protected.

      Haptoglobin (Hp) is a protein produced predominately by the liver . In humans two types of
      genes for Hp exist (1 and 2) with possible combinations of these two genes- 1-1, 1-2, or 2-2.
      The Hp 2 gene is believed to have arisen from the Hp 1 gene in human evolution. Subsequently
      the prevalence of the Hp 2 allele has spread throughout the world, probably as a result of
      its ability to provide a selective advantage against infectious disease. The Hp 1-2
      combination is a very common one. In most western countries, the prevalence of the Hp
      genotypes is 16% Hp 1-1, 36% Hp 2-2 and 48% Hp 2-1.

      The Hp gene form has been shown to be associated with disease. Specifically, Hp phenotypes
      have been found to affect propensity to atherosclerosis in Diabetic individuals. There have
      been several studies suggesting that the Hp 2-2 phenotype is associated with a protection
      against infectious complications.

      In view of the importance of respiratory infections on COPD exacerbations, and of the gained
      knowledge of Haptoglobin subtypes on propensity to infection, we propose to investigate
      whether Haptoglobin subtypes are in correlation with the "frequent exacerbator" phenotype of
      COPD. We postulate that, since people with Hp 1-1 are more prone to infection, the frequency
      of the Hp 1-1 phenotype will be higher in "frequent exacerbators" of COPD than in "non-
      exacerbators".

      To test our hypothesis we propose to determine Hp phenotype in two groups of COPD patients:
      one with frequent exacerbations and one with no exacerbations, and compare the relative
      frequency of the 1-1 phenotype in the two groups.
    

Detailed description: 
      Laboratory Procedures:

      Overview:

      3 ml of Serum will be collected and stored at 4 degrees Celsius (up to one week) or minus 20
      degrees Celsius until delivery to the laboratory.

      Haptoglobin phenotype will be determined at the Department of Anatomy and Cell Biology, the
      B. Rappaport Faculty of Medicine, 1 Efron st. Haifa, Israel. The method for phenotypin Hp is
      protein gel electrophoresis.

      Serum remaining will be kept frozen in minus 70 degrees Celsius for 5 years in a freezer
      shelf dedicated for the Pulmonology Institute at the Serology Laboratory in Carmel Medical
      Center for future analysis of proteins.

      Haptoglobin Electrophoresis:

      The Haptoglobin protein is separated based on size. Hp 2-2 is the largest therefore was
      expected to travel the shortest distance in the gel and Hp 1-1 is the smallest and therefore
      would travel the farthest down the gel. The gel that is used for the electrophoresis was a
      Polyacrylamide gel. Two concentrations are used in order to provide loading and separating
      conditions.

      The lower gel is a Polyacrilamide gel that contains 10.8 ml 1M Tris pH 8.8, 3.55 ml of
      Acrylamide 40%, 11.5 ml of dH2O, 225 μl of Ammonium persulfate (APS) 10%, and 18 μl of
      tetramethylethylenediamine (TEMED). The upper gel is a Polyacrylamide gel that contains 937.5
      μl of 1M Tris pH 6.8, 787.5 μl of Acrylamide 40%, 5.7 ml of dH2O, 75 μl of APS 10%, and 7.5
      μl of TEMED.

      The lower gel is poured in between two glass plates and left for 30 minutes to solidify. Once
      lower gel is solidified upper gel is poured on top of it, and left for 30 min to solidify.
      Wells comb placed superiorly to upper gel in order to create wells for sample placement.

      The sample for each patient is prepared using 10 μl of the serum, 2 μl of Hemoglobin, and 12
      μl of a commercial loading buffer. The sample is then carefully placed in the prepared wells
      using a pipette. 4 wells are reserved for 3 controls of Hp 2-2, Hp 2-1, and Hp 1-1 followed
      medially by an empty well.

      The western blot analysis is run in a Protean II xi cell by BIO-RAD. The apparatus is
      assembled and an electrophoresis running buffer is added. The running buffer contains 15.1 g
      of Tris, 72 g of Glycine, and 1000 ml of DDW. The running buffer was then placed in the upper
      and lower reservoir of the Protean II. The electrophoresis was run at 240 Volts for 3 hrs.

      Once the samples are finished running, the gel is removed from between the glass plates. The
      gel was then stained using a color staining solution. The Solution was made using 40 mg of
      3,3'-5,5' tetramethyl benzidine and 20 ml of methanol. This was left to mix for 15 minutes.
      Next 2 ml of dimethyl sulfoxide is added and left to mix for 5 min. This was followed by the
      addition of 40 ml of acetic acid 5% which is made of 2 ml AA and 38 ml of DDW. Next 40 mg of
      potassium fericyanide dissolved in 4 ml of DDW are added. Finally 600 μl of H2O2 is added to
      the staining solution and left to mix for 10 min.

      Once the staining solution is ready, the gel is placed in a 22 cm by 32 cm tray and the
      staining solution is poured over the gel. The tray is then placed on a shaker for 25 minutes.
      Once the Gel is finished being stained it is legible. Nevertheless, the gel is photographed
      for image enhancement and data storage. The gel is removed from the tray and placed in an
      Image Analyzer (Imagequant LAS 4000) for imaging, or Canon powershot sx40 hs digital camera.

      Data Tabulation:

      The data - the Haptoglobin phenotype with corresponding IDs - is gathered and tabulated into
      a Microsoft excel table.
    

Observational model: Case-Control
Time perspective: Retrospective
Sampling method: Probability Sample
Study population: 
        Patients with COPD
      
Biospecimen description: 
      3 ml of Serum will be collected from each patient
    
Keywords: COPD; Exacerbation; Haptoglobin