ANK-dependent ATP Efflux Causes Calcium Pyrophosphate Deposition in Cartilage
Phase: EARLY_PHASE1
Conditions: Calcium Pyrophosphate Deposition Disease
Sponsor: VA Office of Research and Development

 
 
 
[Year] 
Effect of probenecid on 
synovial fluid ATP levels 
in CPPD 
PROTOCOL AND  STATISTICAL ANALYSIS PLAN: NCT02243631 
FEBRUARY 28, 2022 

 
Study protocol 
 
1. Subjects diagnosed with CPDD according to the McCarty-Ryan criteria are 
identified through the rheumatolgy clinical practice.   
2. Inclusion/Exclusion criteria are met.  
3. Subjects are interested in participating in the study and sign an informed consent 
form and HIPPA.  
4. The affected joint is aspirated for 1 cc of synovial fluid, which is immediately 
placed on ice and sent to the laboratory for analysis of ATP levels 
5. The subject is randomized to receive no additional treatment or to receive 2 
grams/day of probenecid given in two divided doses.  
6. The subject is called daily to monitor symptoms in the affected joint and any side 
effects.  
7. The subject is seen again by the study team on day 6.  Another 1 ml sample of 
synovial fluid is removed from the affected joint and sent to the laboratory for 
ATP analysis.  The subject is then offered an intra-articular corticosteroid 
injection in the affected joint.  
8. This completes the study.  
 
ATP measurement in Synovial Fluid 
 
Basal Salt Solution pH 7.5 
 
 
 
 
 
 
130 mM NaCl  
5 mM KCl 
 
1.5 mM CaCl2  
 
 
 
 
 
 
 
 
1 mM MgCl2  
 
 
 
 
 
 
 
 
 
 
5 mM Glucose 
0.1% BSA 
 
 
 
 
 
 
 
 
 
 
Mix and then check pH, adjust with NaOH 
 
ATP substrate (FL-AAM from Sigma):  Add 5 mL water to vial according to manufacturer’s 
directions. 
 
For standard curve: dilute ATP substrate in salt solution in a ratio of 40% substrate and 60% salt 
solution.  Add 25 l/well for the standard curve. 
 
Standard curve uses the following concentrations of ATP. 
(1 nM, 2.5 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM) 
 
100 l of salt solution is added to the first well to serve as the zero. 
100 l of each of the concentrations of ATP  

 
 
To Measure the synovial fluid (SF) :  100 l of SF (fresh or in citrate) is added to each of 3 wells 
and then 4 l of the stock ATP substrate is added to the wells.  Tap the plate on the counter to 
mix then read immediately in a plate reader set for luminescence. 
 
N.B. Synovial fluid needs to be measured within 1 hour of collection to avoid loss of ATP. 
 
 
 
Statistical Analysis Plan  
The primary outcome will be ATP levels measured from extracted synovial fluid. The two 
treatment groups include those who received probenecid and those with no intervention.  A 
baseline measurement will be taken at the beginning of the study period and another 
measurement taken at 7 days.  Each participant will serve as their own control.  A delta will be 
calculated (7-day ATP level – baseline ATP level) for each participant and a non-parametric 
Mann-Whitney t-test will be performed.  ATP levels commonly require non-parametric analysis 
as it tends not to be normally distributed. The mean change between baseline and the 7-day 
timepoint along with the corresponding 95% confidence interval will be presented.   
A Wilcoxon matched pairs t-test will be used to assess whether the intervention produced a 
statistical difference between the two timepoints with probenecid treatment.