Our overall objective is to develop an R01 grant under the recently released RFA (PA-15-037 Diabetes and Cardiovascular Disease in Older Adults) to examine the association between haptoglobin (Hp) phenotype and CVD risk (incident CHD [MI or revascularization], or stroke) in postmenopausal women with a history of diagnosed treated diabetes comprised of participants in the WHIOS.
In order to develop a competitive larger grant proposal, we need to better understand our ability to measure the Hp phenotypic frequencies (i.e., Hp 2,2; 2,1; and 1,1) in stored plasma samples among WHI women, and using the observed frequencies, calculate statistical power to support specified study hypotheses. Therefore, we propose to conduct a feasibility study in a subsample of the CVD cases and noncases to obtain critically needed information for our R01 submission. The following aims that will guide proposed feasibility study are: (1) to obtain pilot data on our ability to collaborate with Dr. Levy's laboratory to measure the haptoglobin (Hp) phenotypic frequency [aims 1-3] and; (2) to use the observed Hp frequencies in calculating statistical power for the larger grant proposal [aim 4]. Specifically, our aims are as follows:
Determine the frequency of Hp 2,2; 2,1; and 1,1 among 100 diabetic women who have experienced incident coronary heart disease or revascularization following baseline enrollment in WHI;
Determine the frequency of Hp 2,2; 2,1; and 1,1 among 100 diabetic women who have experienced incident stroke following baseline enrollment in WHI;
Determine the frequency of Hp 2,2; 2,1; and 1,1 among a) 100 diabetic women who have not experienced CHD or stroke; and b) 100 nondiabetic women who have not experienced CHD or stroke; and
Use the observed Hp frequencies to calculate statistical power for specified hypothesis in the larger grant proposal.
Except for the Hp phenotyping, the data for this proposal (and our planned R01) have already been collected with the biological samples resting with the Coordinating Center. Our approach would be to identify a random subsample of CVD cases (100 CHD; 100 stroke) with history of diabetes, as well as a random subsample of noncases with diabetes (100) and noncases without known diabetes (100), and retrieve their stored plasma samples. The samples) would then be sent overnight, blinded to Dr Levy's laboratory) for Hp phenotyping. The results would then be sent to the CC for unblinding and then onto SUNY Buffalo for analyses. Analyses would consist of examination of the Hp phenotype frequency against Hardy-Weinberg equilibrium according to a chi-square test in the various groups followed by examination of the Hp frequencies and risk of the event(s) in question while controlling for covariates of interest using a logistic modeling approach. This will provide excellent preliminary data that will strengthen an ancillary study application.
All 440 samples were analyzed using an Hp type ELISA kit (1) as follows: All incubations were done at room temperature while rotating at 750 rpm. All washes were done by filling the wells completely with PBS containing 0.1% PBS, removing the wash buffer by vacuum aspiration, and tapping the plate onto absorbent paper. Serum samples were diluted 1:10 (15 ul serum was added to 135ul sample dilution buffer in a 96 well microtiter dish). Standards were also added to the microtiter dish (150 ul of sample buffer only and 150 ul each Hp 1-1, Hp 2-1, and Hp 2-2 standard sera supplied with the kit). Diluted samples and standards (100 ul) were transferred to a 96 well plate that had been previously coated with antibody to Hp. The plate was incubated and washed, then horseradish peroxidase conjugated antibody to Hp was added (100 ul/well). Following incubation and washing TMB substrate was added for 20 min. After incubation the reaction was stopped with 100 ul Stop solution and the OD at 450 nm recorded using and ELISA reader.
Samples with an OD of 0.2 or less were scored as Hp 1-1 by ELISA. An Hp 2-1/Hp 2-2 cutoff is calculated by multiplying the OD obtained with the Hp 2-2 standard by 0.6. Samples with an OD greater than 0.2 and less than or equal to the Hp 2-1/Hp 2-2 cutoff were scored as Hp 2-1. Samples greater than the Hp 2-1/Hp 2-2 cutoff were scored as Hp 2-2.
Any samples (21 out of the 440 WHI samples) with an OD 450 between 0.1 and 0.2 or within 10% of either cutoff were reanalyzed by gel electrophoresis (2). In addition for QC an additional 22 samples were randomly selected to be additionally analyzed by gel electrophoresis. Briefly, 2 ul of serum are mixed with 2 ul of Hb (10 mg/ml) and 18 ul 1x sample buffer and electrophoresed in a non-denaturing polyacrylamide mini gel. Known sera from Hp 1-1, Hp 2-1, and Hp 2-2 individuals are also applied to the gel. Following electrophoresis the gel is incubated in a solution containing a colorometric substrate (TMB). The Hp/Hb complexes oxidize the substrate to produce a greenish precipitate. Each Hp type has a distinct banding pattern with 4 possible Hp types assigned: Hp 1-1, Hp 2-1m (a type intermediate between Hp 1-1 and Hp 2-1), Hp 2-1 and Hp 2-2. Note that the Hp 2-1m type can only be assigned by gel electrophoresis and falls in the ELISA between Hp 1-1/2-1. This Hp 2-1m type is relatively rare among Caucasians but more common in African Americans. The final Hp type assigned to the samples when there was a discrepancy between the gel and the ELISA. There were 13 samples assigned Hp 2-1m by gel (these samples fell out in the borderline region between Hp 1-1 and Hp 2-1 by ELISA as expected). There was one sample (#135 by our numbering scheme) which by ELISA was read as Hp 2-2 but by electrophoresis was read as Hp 2-1 and therefore reported with final result of Hp 2-1. There were no other discrepancies between the two methods.
There remains considerable uncertainty of the role of Hp phenotypes in CVD. Extant studies have several limitations including: 1) combining stroke with CHD; 2) relatively small numbers of events leading to a lack of precision and subsequent wide confidence limits; and 3) whether assessing HP phenotypes can contribute to a finer stratification of risk over and above traditional risk factors. This feasibility study would provide fertile ground for more complete testing of the overall hypothesis that Hp 2,2 constitutes a risk factor for CHD while Hp 1,1 may be a risk factor for stroke.