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INTRODUCTION
Address correspondence to: A. Mastaloudis, Pharmanex Research Institute, 75 W. Center Street, Provo, UT 84601. E-mail: amastaloudis@pharmanex.com
Data presented here have been published previously in abstract form in Zidichouski JA, Poole SJ, Gellermann W, Smidt CR: Clinical validation of a novel Raman
spectroscopic technology to non-invasively assess carotenoid status in humans. J Am Coll Nutr 23:A468, 2004.
Dr. Zidichouski is now with the Institute for Nutrisciences and Health, National Research Council of Canada.
Dr. Reading is Professor Emeritus at the Department of Family and Preventive Medicine, University of Utah School of Medicine.
Dr. Smidt is now at Shaklee Corporation, Pleasanton, California.
This study was funded by the Pharmanex Research Institute, Pharmanex LLC, Provo, Utah.
Author Contributions: Jeffrey A. Zidichouski was responsible for study design, subject recruitment, data collection, and analysis and preparation and revision of the
manuscript; Angela Mastaloudis contributed to the statistical analyses and was responsible for the preparation and revision of the manuscript; Stephen J. Poole contributed
to the subject recruitment, data collection, and data analyses; James C. Reading contributed to the statistical analyses; Carsten R. Smidt contributed to the study design,
offered expertise in carotenoids and Raman spectroscopy, and contributed to manuscript revisions. A.M. and S.J.P. are currently employed by Pharmanex Research
Institute (Pharmanex, LLC). J.A.Z. and C.R.S. were employed by Pharmanex Research Institute (Pharmanex, LLC) at the time the study was conducted.
Journal of the American College of Nutrition, Vol. 28, No. 6, 687693 (2009)
Published by the American College of Nutrition
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Protocol
Over the course of 8 days, each subject visited the lab for
blood sample collection and RS determination 3 times, with at
least 48 hours between each sample collection. Each day, a 10ml blood sample was collected from an antecubital arm vein
into untreated glass vacutubes; all were fasting, morning blood
draws. Within 15 minutes of blood sampling, skin carotenoid
concentrations in the palm of the hand were determined using
RS technology. All data were obtained over a 3-month period
and without any changes to study personnel.
Statistical Analysis
Descriptive statistics (means, medians, standard deviations,
etc.) were calculated and statistical tests were conducted to
compare and contrast the RS and HPLC methodologies of
determining carotenoid nutritional status. All data are presented as means 6 standard deviations.
Criterion validity was used to validate the RS methodology
by comparing it to serum carotenoid quantification by HPLC.
Based on individual data for each subject (n 5 372), scatter
plots, lines of best fit (with 95% confidence curves for the
fitted line), and Pearson correlation coefficients were generated for total serum carotenoid concentration versus the sameday RS skin carotenoid determination for each of the following 3 test periods: serum 1 vs. RS 1 (sample 1), serum 2 vs.
RS 2 (sample 2), and serum 3 vs. RS 3 (sample 3). In order to
generate an overall estimate of the line of best fit, in addition
tothe 3 (1 for each serum sample) above analyses, an analysis
of covariance (ANCOVA) was performed using all 3 samples
(1116 observations) and modeling subject ID as a main effect.
To evaluate the consistency between methods, the intraindividual variability of both the RS and the HPLC methods
was determined. Pooled estimates of variance (from a 1-way
analysis of variance [ANOVA]) of the 3 RS and serum
measurements across individuals were calculated. Variance of
the pooled estimates was compared with an F-test. For each
subject, the standard deviation of each RS determination and
day-matched serum sample was calculated and the population
of standard deviations was used to perform a Wilcoxon test for
differences in medians between the RS count and serum HPLC
analysis. Statistics were calculated using JMP Statistical
Discovery Software (SAS Institute Inc, Cary, NC).
RESULTS
Of the 391 volunteers, 5 were excluded due to pregnancy or
breastfeeding and 14 were withheld from the final analysis due
to incomplete datasets. Data presented here are for the 372
subjects (95% of enrolled subjects) who completed the study,
providing complete skin and blood sample datasets. Of these,
199 were men and 173 were women; the average age was 33.4
6 10.0 years (range 1868 years). There was no significant
Validity
Correlation between Skin Carotenoids by RS and Total
Serum Carotenoid Concentrations by HPLC. For the first
measurement, the mean RS value was 20,095 6 6439 (range 9137
49,666 counts) and the mean total serum carotenoid concentration
was 1.08 6 0.51 mg/ml (range 0.213.74 mg/ml). A positive linear
correlation between RS and total serum carotenoid concentration
was observed (R 5 0.82; r2 5 0.67; p , 0.001; Fig. 1A).
For the second sampling, mean RS skin carotenoid count
was 20,178 6 6367 (range 862448,441 counts) and mean
total serum carotenoid concentration was 1.08 6 0.50 (range
0.233.97 mg/ml). A direct correlation between RS and total
serum carotenoid concentration was observed (R 5 0.80, r2 5
0.64; p , 0.001; Fig. 1B).
In the third and final sampling, mean RS skin carotenoid
count was 20,034 6 6352 (range 788849,262 counts) and the
mean total serum carotenoid concentration was 1.14 6 0.52
(range 0.224.41 mg/ml). Consistent with samplings 1 and 2, a
strong positive correlation between the 2 measures was
observed (R 5 0.81, r2 5 0.65; p , 0.001; Fig. 1C).
A linear regression was performed with RS as the
dependent variable and serum concentration as the independent
variable. The results of the best fit lines for the 3 samples were
sample 1: slope 0.000066, intercept 20.24 (y 5 20.24 +
0.000066x); sample 2: slope 0.000063, intercept 20.19 (y 5
20.19 + 0.000063x); and sample 3: slope 0.000066, intercept
20.19 (y 5 20.19 + 0.000066x), where x 5 Raman intensity
counts and y 5 serum carotenoid concentration.
The composite correlation based on the overall estimate of
the line of best fit from ANCOVA, using all 3 samples (n 5
1116) and modeling subject ID as a main effect, was: slope
0.000064 with intercept 20.09 (20.09 + 0.000064x) with a
Pearson correlation of R 5 0.81 (r2 5 0.66; p , 0.001).
Reliability
Pooled Estimate of Variance. Based on ANOVA, the RS
skin carotenoid pooled estimate of standard deviation was
0.095 compared to a pooled estimate of standard deviation of
0.104 for serum carotenoids by HPLC. Results demonstrate
that the RS skin carotenoid method resulted in less variance
over the 3 tests than did serum carotenoids quantified by the
HPLC method (skin carotenoid standard deviation 9.5% of the
mean compared to 10.4% for serum carotenoids; p , 0.03).
Wilcoxon Test. To further investigate the variance of the 2
methods, for each individual, the standard deviation of each set
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Fig. 1. RS skin carotenoid concentrations were significantly correlated with total serum carotenoid concentrations on all 3 days; (A) sample 1, r2 5
0.67 (p , 0.001); (B) sample 2, r2 5 0.64 (p , 0.001); (C) sample 3, r2 5 0.65 (p , 0.001). N Represents data for individual subjects; R 5
Pearson correlation.
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Variability
The mean RS value was 20,102 6 6381 counts with an
intrasubject coefficient of variation of 8.2% for 3 measurements over 8 days. The mean HPLC carotenoid concentration
for this group was 1.102 6 0.514 mg/ml, with an intrasubject
coefficient of variation of 8.9% for 3 measurements over
8 days.
DISCUSSION
Results of the present study demonstrate that RS-based
technology is a valid and reliable technique to rapidly and
noninvasively assess carotenoid status in humans. This is the
first large-scale clinical study to systematically validate the RS
method to accurately assess carotenoid status in situ in living
human skin. The concurrent validity technique was used to
directly compare the RS method against the HPLC analysis
method, the current gold standard for determination of
carotenoid status in human subjects.
Originally, the RS methodology was developed in an effort
to detect carotenoid pigments in the macula of the eye by
researchers studying the relationship between carotenoids and
macular degeneration [17]. While this research remains active
[1823], development of the technology has been expanded to
the measurement of carotenoids in human skin [10,11,13,14].
A fast, reliable, and noninvasive technique to assess carotenoids in human skin offers enormous potential for evaluating
and tracking carotenoid status in large populations.
Previously, Hata et al. [14] validated the RS technique
against HPLC analysis of skin carotenoids in abdominal skin
obtained from a tissue bank. A positive correlation between
skin carotenoids measured by HPLC and by RS technique was
observed. However, no correlation coefficients were reported,
likely due to limited sample sizes. The authors reported that RS
methodology accurately measures carotenoid concentration in
human skin with a high degree of reproducibility [14]. In our
initial pilot work, we observed a positive correlation between
total skin carotenoids using RS methodology and total serum
carotenoid concentration measured by HPLC (R 5 0.78, p ,
0.001) in 104 subjects [15]. Consistent with this research, in
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CONCLUSIONS
Based on the present study, the RS method is a valid and
reliable technique to measure carotenoids in human skin in
vivo. There is a strong correlation between skin and serum
carotenoid concentrations. Due to its noninvasive nature and
low variability, the RS-based method is an attractive tool for
assessing carotenoid status in humans and appears to be
superior to the costly, more invasive blood carotenoid HPLC
method.
ACKNOWLEDGMENTS
The authors are grateful to Neil Craft, PhD, and his staff at
Craft Technologies (Wilson, NC) for their expertise in HPLC
analysis of serum carotenoids.
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