2. The Effects of Dietary Caffeine on Blood Alkaline Phosphatase Levels in Pre-Menopausal Caucasian Women Jonathan Buchanan Biology 493

3. Definitions  Alkaline Phosphatase A dimeric enzyme released by the osteoblast cells in bones that increases local concentrations of inorganic phosphates or active collagen fibers in such a way as to deposit calcium salts on the bone matrix during osteogenesis.  Caffeine ( 1,3,7Trimethylxanthine ) A planar two-ring molecule with the chemical formula of C 8 H 16 N 4 O 2 found in coffee, chocolate, tea, and some soft drinks.

4. Review of Literature  Osteoporosis is a common condition in post-menopausal women (Nakamoto & Shayne 1986).  Lowered alkaline phosphatase (AP) activity in the bone is considered to be one of the factors leading to osteoporosis (Bauer et al. 1977).  Caffeine is thought to have a detrimental effect on bone ossification (Heaney & Recker 1982).  Caffeine lowers AP levels in rat tissues (Barone et al. 1993).  Gender contributed to the effect of caffeine on serum AP levels in rats (Palm et al. 1984).

5. Review of Literature (Continued)  Carbonation in sodas had no effect on urinary calcium levels in patients with calciuria (Heaney & Rafferty 2001).  Young women showed no change in bone density after dietary consumption of non-caffeinated beverages (Smith et al. 1989).  Carbonation has no effect on bone density in older, post- menopausal women (Kim et al. 1997).  Age has no effect on the AP diminishing activity of caffeine (Driscoll et al. 1982).

6. Purpose The purpose of this study was to determine the effects of caffeine, when ingested daily, on serum alkaline phosphatase levels in pre-menopausal females.

7. Methods & Procedures 1.Blood Samples were taken from 16 caffeine-na ï ve Caucasian female college students into SST (Gel and Clot Activator) Tubes using sterile technique. 2.Each student was administered unmarked soda bottles & instructed to ingest one daily for a two week period of time. They were not informed of the soda ’ s caffeine content.

8. Subject #kgsmgC/kgBW 163.5041.5747 261.2361.6330 363.5041.5747 468.041.4697 563.5041.5747 668.041.4697 768.041.4697 861.2361.6330 Mean1.55 Max1.63 Min1.47 StDev0.07 Caffeine Content The amount of caffeine in Pepsicola TM ranges from 55 to 65 mg per 12 ounce can (Bunker et al. 1979). Therefore, each subject ’ s daily caffeine consumption was ~100mg/day. The amounts of caffeine in mg Caffeine/kg body weight for each person tested were determined.

9. Procedure (Cont.) 3. Blood samples were centrifuged at 3400 rpm for 10 min. 4. Supernatant was transferred into numbered test tubes with a 100 μ L micropipette.

10. 5.Samples were frozen at -4 ⁰ C for storage. 6. Samples were sent to Diagnostic Labs for alkaline phosphatase level determination. Procedure (cont.)

11. Results Caffeinated Pepsicola TM Non-caffeinated Pepsicola TM

12. Results (cont.) Paired T-Test and Confidence Interval Paired for placebo before and placebo after NMeanStDevSE Mean p before 872.7518.51 6.55 p after865.6212.85 4.54 Difference8 7.1310.44 3.69 95% CI for mean difference: (-1.61, 15.86) T-Test of mean difference = 0 (vs not = 0): T-Value = 1.93 P-Value = 0.095 Paired for caffeine before and caffeine after NMeanStDevSE Mean c before847.2511.35 4.01 c after855.2513.14 4.65 Difference8 -8.00 7.05 2.49 95% CI for mean difference: (-13.90, -2.10) T-Test of mean difference = 0 (vs not = 0): T-Value = -3.21 P-Value = 0.015 Non-caffeinatedCaffeinated Before After * - Significant * Statistical analysis via a repeated measure t-test revealed a significant change in alkaline phosphatase levels following the two week study period. Apparent changes in control AP levels were not statistically significant.

13. Results (Cont.) When analyzed against a statistical regression, data collected regarding stage in menstruation showed no significant effect of menstrual hormones on caffeine ’ s effect to change AP levels. Analysis of Variance SOURCE DF SS MS F p Regression 1 4.6 4.6 0.03 0.855 Error 14 1875.4 134.0 Total 15 1880.0

14. Discussion  Changes in alkaline phosphatase could be attributed to a response by the osteoblast cells to the demineralization of the bone and subsequent decrease in bone density brought on by lowered calcium.  The discrepancy between the results of this study and those of previous researchers (Barone et al.1977, & Palm et al. 1984) can be attributed to the difference in amounts of caffeine administered to the subjects per kilogram body weight.  The drop in AP in the control group, although not significant, could be attributed to the carbonation content in the sodas, which would be contrary to the results of Smith et al. (1989)

15. Conclusion If small amounts of caffeine (~100mg) ingested daily is sufficient to alter bone demineralization as measured by a change in alkaline phosphatase, women with a familial history of osteoporosis should consider consuming little or no caffeine as part of their diet.

16. Acknowledgments Dr. Randy Day Diagnostic Laboratories William W. “ Wurt ” Edwards Ramona Granat BYUH Faculty & Staff Levi & Shalon Wilmott My sweet wife, Sarah

17. Cola

18. Analysis of Sample Size