The Investigation of X-Ray Diffraction Patterns from Human Hair in Correlation with Dietary Habits

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Savannah Gore and Professor Beverly L. Roeder, Department of Biology

Eating disorders, namely anorexia nervosa (AN) and bulimia nervosa (BN), are a serious problem affecting 1-5% of high school and college age women in the US. Unfortunately, the diagnostic methods used to determine if an individual is suffering from AN or BN are, for the most part, based on fallible self-report data. In an attempt to alleviate this problem, a quantitative, biologically based test able to accurately diagnosis AN 80% of the time using carbon and nitrogen stable isotope fractionation in hair was developed. In an effort to raise the accuracy of this test, I looked at x-ray diffraction patterns from hair to see if changes in the molecular structure reflected the severe malnutrition found in individuals suffering from eating disorders.

My hypothesis was based on previous findings that the molecular structure of α-keratin, the primary protein component of hair, may be altered by diseases affecting metabolism and protein synthesis. Specifically, changes in α-keratin provide distinct x-ray diffraction patterns for breast cancer, colon cancer, diabetes and Alzheimer’s disease. , , , The researchers in these studies hypothesized that the observed structural changes were probably due to upsetting the normal balance of hormones and signaling factor activity, some of which, among other things, regulate hair growth. Since it has also been observed that many chemical balances are altered in anorexic individuals, and it is known that thin, brittle hair is a symptom of anorexia , I thought that perhaps structural changes might also be observed in hairs from anorexic individuals.

To test my hypothesis, I used scalp hairs collected as a part of a larger study investigating the effects of dietary habits and exercise on the stable isotope fractionation in human hair aimed at refining the diagnostic test for AN.1 Experimental samples were collected from a single individual with AN who was enrolled in a treatment program through the study and who volunteered to donated hair. Although ideally it would have been better to run the experiment with samples from multiple individuals with eating disorders, since none of the other subjects desired to participated, I decided to limit my investigations to generating preliminary data which would indicate whether my hypothesis merited further investigation. I randomly chose four hair samples from the larger group of controls in the study to act as controls in this experiment. For three of the controls, two hairs were tested to check for variance across individuals. The remaining control contributed one hair. Three hairs were tested from the AN patient, also to check for variance within a single person. Samples were collected and run by separate investigators in the study and each individual was given an ID number at the time of collection to maintain confidentiality. Approximately a three-centimeter segment was cut from the proximal end of the hair. The segments were then placed in brass mounting pins with the proximal end facing downward and attached with modeling clay, leaving 1-1.5cm above the top of the pin. Thus the hair exposed to the beam was synthesized 2-3 months previous to collection. The data were collected from a each hair sample at room temperature on an APEX II CCD detector system using a Bruker FR-591 rotating Molybdenum radiation source with a wavelength of 0.71073 angstroms. For each sample, the X-raygenerator was set at a 50kV and 60mA power setting, and the diffraction patterns were collected using a 359 degree phi rotation scan with a 30 minute exposure times and a 240mm detector distance. The beam stop was pushed all the way back to increase the amount of low angle scattering observed.

We found that there were two patterns of x-ray diffraction: the first was a clear yellow ring (figure 1) and the other consisted of no discernable ring patterning (figure 2). At this point it is difficult to say whether the separate patterns indicate structural differences due to AN or another factor. Three of the controls exhibited the clear yellow ring pattern in either one or both hairs, while a single control sample did not. All three samples from the anorexic individual had little visible diffraction patterning (figure 2). Although there is some intra-individual variation, generally it seems that normal individuals generate the first ring pattern. However, as mentioned before, one control did not exhibit this pattern, nor did one of the hairs from another control. This then begs the question as to whether those two samples generated an x-ray diffraction pattern similar to the AN hairs because the individual was actually suffering from malnutrition at the time of synthesis. It is also possible that the common pattern could be due to another factor, such as certain hair treatments. With a small group of controls and a single AN hair sample, it is impossible to tell whether the correlation of the distinctive ring pattern with controls and the opposing correlation of little diffraction with AN means anything.

Despite the disparity in the results, this observed correlation does merit further investigation. The results are inconclusive and therefore, while they do not fully support my hypothesis, they do not completely discount it either. Ideally, my work should be repeated with a much larger sample size. Especially, future researchers should attempt to procure more samples from AN and BN patients, which we were unable to do. Furthermore, I would suggest that future research be done on a synchrotron, an instrument similar to the X-ray diffractometer used in this experiment but able to produce images of a higher resolution.

References

  • Hatch KA, Crawford MA, Kunz AW, Thomsen SR, Eggett DL, Nelson ST, Roeder BL (2006) An objective means of diagnosing anorexia nervosa and bulimia nervosa using 15N/14N and 13C/12C rations on hair. Rapid Commun. Mass Spectrom 20: 3367-3373
  • James VJ, Yue DK, McLennan SV (1997) Changes in the molecular structure of hair in insulin-dependent diabetes. Biochem Biophys Res Comm 233: 76-80
  • James V, Kearsley J, Irving T, Amemiya Y, Cookson D (1999) Using hair to screen for breast cancer. Nature 389: 33-34
  • James V (2003) False-positive results in studies of changes in fibre diffraction of hair from patients with breast cancer may not be false. J Natl Cancer Inst 95(2): 170-71
  • James VJ, Richardson JC, Robertson TA, Papadimitriou JM, Dutton NS, Maley MAL, Berstein LM, Lansteva OE, Martins RN (2005) Fibre diffraction of hair can provide a screening test for Alzheimer’s disease: a human and animal model. Med Sci Monit 11(2): CR53-57
  • Mekota AM, Grupe G, Ufer S, Cuntz U (2006) Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa. Rapid Commun. Mass Spectrom. 20:1604-1610
  • Special thanks to Dr. Beverly Roeder and Dr. Kent Hatch for mentoring me in this research, Dr. Diane Spangler for obtaining the AN hair samples, Dr. Steven Thompson for collecting the control samples, Dr. Branton Campbell for counseling me extensively on the physics involved in this project and Dr. Steven Herron for running all the samples on his X-ray diffractometer.