Three females and three males participated in this pilot study. All subjects in this investigation participated in a familiarization session. During the familiarization session, subjects were informed as to the experimental procedures, completed a personal/medical history form, creatine supplementation history form and signed informed consent statements in adherence with the human subject's guidelines of the American College of Sports Medicine. The study was approved by the Ethical Review Committee of the University of Chichester. Subject characteristics are presented in table 1. No subject in this trial was a vegetarian with all subjects reportedly consuming meat in their daily diet.
The study used a cross-over design. Each subject received the three treatments using a latin-square design on three different days, with 7 days allowed between each treatment. Treatments comprised:
A: 5 g Creatine monohydrate (CrM) dissolved in 450 ml of water (control)
B: 6.7 g Tri-creatine citrate (CrC) dissolved in 450 ml of water (isomolar with respect to creatine to A and C)
C: 7.3 g Creatine pyruvate (CrPyr) dissolved in 450 ml of water (isomolar with respect to creatine to A and B).
The creatine monohydrate (Creapure™) contains 88% w/w creatine, creatine pyruvate (Creapure™Pyruvate) contains 60% w/w creatine and 40% w/w pyruvate, and, tri-creatine citrate (Creapure™Citrate) contains 65% w/w creatine. All three were obtained from Degussa AG (Trostberg, Germany) and contained <100 ppm creatinine, whilst dicyandiamide and dihyrdotriazine levels and polymeric pyruvates in CrPyr were not detectable by HPLC.
Two ml blood samples were collected into tubes containing lithium heparin through an antecubital vein, centrifuged and plasma was harvested and stored frozen until analysed. Plasma (500 μl) was extracted with 15 μl of 70% w/w PCA and supernatants after centrifugation were neutralised with a minimum volume of 2N KHCO3 at 4°C. One hundred and sixty one blood samples, from an intended 162, were collected. Data for from the missing sample (subject 1, treatment C, 2 hours) were estimated from the two adjacent points assuming an exponential decrease between 1.5 to 3 hours. Creatine in plasma extracts was analysed photometrically by following the oxidation of NADH at 340 nm in the presence of creatine kinase, pyruvate kinase and lactate dehydrogenase . Pyruvate was analysed using a similar assay but with the addition of lactate dehydrogenase only.
Using a Marquardt algorithm, all kinetics were fitted with the following equation:
C is the plasma creatine concentration minus the basal concentration (μM)
if C ≤ 0, then data were removed
F is the bioavailability
D is the dose (5 g or 38.17 mmol)
V is the volume of distribution
ka is the velocity constant of absorption (first order, h-1)
kel is the velocity constant of elimination (first order, h-1)
t is the time (h)
No weight was applied to data (weight = 1) and the initial parameters values were estimated using a Simplex algorithm. This equation corresponds to a one-open compartmental model. The area under the curve (AUC) from zero to infinity was estimated by non compartmental model
Results are shown as means together with standard deviation. Primary and derived variables were analysed by repeated measures ANOVA. Where a significant effect of treatment was indicated, data were further compared using a Bonferroni post-hoc test. The threshold for significance was set at p < 0.05.