Oral Creatine Supplements Lower Plasma Homocysteine Concentrations in Humans

Clinical Laboratory Science, Spring 2004 by Korzun, William J

To begin the study, each subject was given a bottle of multi-vitamins and was instructed to take one tablet every day for the duration of the study. They were also instructed to refrain from using creatine supplements until further notice. After four weeks, a fasting blood sample was collected by venipuncture and processed as described below. Each subject then collected and delivered a 24-hour urine specimen. The subjects were randomly assigned to either a control group (C) (n = 10) or an experimental group (EX) (n = 9), and their 24-hour creatinine excretion rates were measured. During the next four weeks, those in EX ingested, in addition to the multivitamins, a single daily dose of creatine monohydrate, added to the room temperature or chilled beverage of their choosing, equal to twice their daily creatinine excretion on a molar basis. Each subject in EX was provided a bottle of creatine monohydrate and a plastic scoop with a calibration mark to indicate the daily dose of creatine monohydrate for that subject. The daily dose of creatine monohydrate for each subject in EX was determined with the following formula:

(Daily dose of creatine monhydrate) = (2) × (24-hour creatine excretion rate) × (1.319)

The 24-hour creatinine excretion is expressed in g/24 hrs., the factor of 1.319 represents the ratio of the molecular weight of creatine monohydrate (149g/mole) to the molecular weight of creatinine (113 g/mole), and the factor of 2 is used to insure an excess of creatine ingestion relative to metabolic demand. For example, subject 001 had a creatinine excretion rate of 1.17 g/24 hours, and was given a scoop calibrated for 3.1 g of creatine monohydrate. The dose of creatine ingested by subjects in EX ranged from 2.1 to 5.5 grams per day. During the second four weeks, those in C continued to take only the multi-vitamins. A second fasting blood sample was then collected from each subject at the end of the second four week period.

After an overnight fast, blood samples were collected by venipuncture into three commercially available evacuated tubes. One tube contained tripotassium EDTA, one contained lithium heparin, and the third contained no additive. Immediately after completion of the venipuncture, 100 µL of the EDTA-whole blood was mixed with 1.0 mL of folate lysis reagent (Abbott product # 9C13-60) containing ascorbic acid and guanidine hydrochloride, incubated at room temperature in the dark for 90 minutes, and then stored at -70 °C. The remainder of the EDTA specimen was centriftiged at 1500 x g at 4 °C for ten minutes, and the plasma then aliquoted and stored at -70 °C. The subject's hematocrit was determined from the heparinized sample. The additive-free blood was allowed to clot in the dark at room temperature for 30 minutes, centrifuged at 1500 x g at 4 °C for ten minutes, and the serum aliquoted and stored at -70 °C. Upon receipt, the 24-hour urine samples were mixed, the volumes measured, and aliquots were stored at -70 °C.

All frozen samples for a given analyte were analyzed on the same day, immediately after thawing at room temperature and mixing. All samples from a given subject were analyzed in the same run. Immediately prior to analysis, thawed hemolysates for erythrocyte folate levels, and urines for creatinine measurement were diluted with the protein diluents recommended by the manufacturers of the analyzers with which those tests were performed.