Carotenoids are thought to play an important role in health and disease state of human tissue. Based on their antioxidant and optical filtering functions,1 they may protect against various cancers,23.4.–5 cardiovascular disease,6 macular degeneration,7 and all-cause mortality.8 In human skin, the composite carotenoid score can serve as an objective biomarker for fruit and vegetable intakes9 and maybe even as an integrative biomarker of health,10 since many dietary carotenoid species are accumulated in this tissue. These include, in order of decreasing concentrations, -carotene, lycopene, lutein, -carotene, -cryptoxanthin, and zeaxanthin. The skin carotenoid levels can be rapidly and noninvasively assessed with optical methods in clinical as well as in field settings, making it possible to investigate correlations between fruit and vegetable intakes and health outcomes in large populations.10
In this article, we explore the presence of carotenoids and their potential optical detection in human bone and surrounding tissue. Bone is a living, dynamic, connective tissue which protects various organs, provides rigidity to the human skeleton, produces all blood cells, generates osteocalcin, and stores minerals for key biochemical processes. The optimal health and homeostasis of bone involve a dynamic balance between bone formation and resorption as well as a multitude of biochemical reactions necessary for life. This balance requires an intimate relationship with the environment, particularly as it relates to the food intake.
Indirect previous studies have already hinted at a potential beneficial role of carotenoids in bone health. Using bone tissue cultures in vitro, -cryptoxanthin has been linked to the stimulation of bone-forming osteoblasts and to the inhibition of osteoclastic bone resorption, thereby increasing bone mass.11 Epidemiological studies have suggested that high intakes of vegetables and fruits reduce the risk of osteoporosis, that carotenoid antioxidants are beneficial micronutrients for the maintenance of normal bone metabolism, that serum levels are associated with bone mineral density in postmenopausal women,12 and that total carotenoid and lycopene intakes have a protective effect on hip fracture.13
Carotenoids absorb strongly in the blue-green wavelength region, and at high tissue concentrations, they are recognizable by the yellowish color they impart on the living human tissue. Long-term visual observations of the appearance of bone and connective tissue, exposed during open-knee implant surgery, by one of the authors (TDR) suggest that the integration/ingrowth capabilities of spongy femoral and tibial bones following total knee joint replacement correlate with the strength of a yellowish coloration of the bone and surrounding fatty tissue, which can be speculated to be due to the presence of carotenoids. Potentially, a strong correlation could exist between tissue carotenoid levels, healing responses after surgeries, and general bone health.
Examples for the differing visual appearances of flat femoral spongy bone surfaces prepared for the insertion of artificial knee joints in two patients are shown in Fig. 1. The photograph in the upper panel shows a femoral bone surface with a relatively healthy internal bone structure and exhibits a strong yellow coloration. The photograph in the lower panel shows the cut surface of the distal femur having various pathologies (compromised structure, cysts, inflammation, and areas of locally reduced densities) and exhibits a relatively pale coloration. Both photographs were obtained during surgery under similar lighting and recording conditions. The difference in the yellowness of the exposed bone and fatty tissue in the two patients suggests a significant difference in tissue carotenoid levels.
Motivated by these observations, we investigated the composition and relative abundance of carotenoids in knee tissue, demonstrated the possibility to detect the composite carotenoid score optically with resonance Raman and reflection spectroscopies, and discussed skin carotenoid measurements as a potential surrogate marker for bone carotenoid status.
Resonance Raman Spectroscopy
To prove the existence of carotenoids in bone and surrounding tissue, we first employed resonance Raman spectroscopy (RRS), which has emerged as a highly molecule-specific methodology for the noninvasive optical detection of carotenoids in the living human retina and in skin.1415.–16 In bone research, Raman spectroscopy has already proven to be a valuable tool for the measurement of mineral composition.17,18
Wedge-shaped bone samples biopsied during total knee arthroplasty were prepared with at least one flat surface for optical interfacing. All samples were obtained from a standard bone cut (resection) at the postero-lateral femoral condyle during total knee implant surgery while under strict tourniquet control of bleeding. Each sample was placed into a sterile plastic cup, capped, and refrigerated within 30 to 60 min of excision. The biopsy site was selected because it appears uninvolved in the osteoarthritic degradation affecting diseased areas of the knee joint, namely the medial weight-bearing compartment and the patella-femoral compartment. As a consequence, it reflects more nearly the undiseased bone condition. In approximately 50% of all cases, samples were obtained from athletically active patients suffering from posttraumatic or secondary osteoarthritis.
After short storage at ice temperature, samples were warmed up to room temperature and placed in direct contact with the window of the light delivery and collection module of the RRS instrument. As an excitation source, an argon laser was used that generated power at 488 nm over a beam spot size of at the bone surface. The RRS spectra were recorded with a CCD-coupled grating spectrograph and displayed on a computer monitor. Representative bone spectra are shown in Fig. 2, where Fig. 2(a) shows the raw bone emission spectrum as a strong, spectrally broad, “autofluorescence” response with superimposed, weak, sharp, Raman lines, and Fig. 2(b) shows the isolated carotenoid RRS response obtained after fitting the smooth fluorescence background with a fourth-order polynomial, subtracting it from trace Fig. 2(a) and expanding it vertically on the intensity axis. All three major RRS lines characteristic for carotenoids are visible. These includes the Stokes lines at , corresponding to the rocking motion of the molecules’ methyl side groups, the line at , corresponding to the single-bond stretch vibration, and the strongest line at , corresponding to the double-bond stretch vibration.14 The intensity of the latter was used as a quantitative measure of all carotenoid subspecies present in the tissue, since all subspecies contribute to the Raman intensity through their common backbone vibration.14
In Fig. 3, we show the RRS results for the trabecular femoral bone biopsies of 33 patients as a histogram. Carotenoid RRS levels vary significantly between samples/patients with a maximal-observed sixfold difference between subjects. Qualitatively, this intersubject variation is similar to the carotenoid concentration variations found in other tissues, such as in the macular region of the human retina or in skin, and is very likely correlated also, in this tissue, to differences in dietary habits, in uptake, in lifestyle factors, such as smoking, and other determinants.10
The availability of excised tissue samples offered the possibility to establish a direct correlation between RRS-derived bone carotenoid levels and concentrations of carotenoid extracts. For this purpose, we ground up small bone fragments with a pestle in a porcelain mortar under acetone, transferred the obtained fine slurry to a 0.5-in. diameter glass test tube filled with 4-ml acetone, and let the tube sit at room temperature for 30 min to allow heavier particles to precipitate at the bottom. Subsequently, 2 ml of the extract was collected from the top of the test tube with a syringe and placed in a fused silica cell for direct absorption measurements.
A typical absorption spectrum measured through such a bone carotenoid extract with 1-cm path length is shown in Fig. 4 and compared with the spectrum of a pure -carotene solution measured with the same instrument. The raw absorption spectrum features a high-scattering background, which is due to the high concentration of lipids extracted from the bone marrow. After background subtraction, the remaining spectrum features an absorption band shifted by about 15 nm to shorter wavelengths with respect to pure -carotene. In spite of the minor differences, these absorption results clearly confirm the presence of carotenoids in the extract and correspondingly in the bone samples. From the absorption strength of 0.024 optical density units at 435 nm for the sample of Fig. 4(a), measured over an optical path length of 1 cm, a total carotenoid concentration of in the extract and in the tissue can be estimated. Here, we approximate the extinction coefficient for bone carotenoids with that for -carotene in petroleum ether solution, i.e., ,19 then use the expression
In agreement with the yellowish appearance of the bone samples, their concentrations appear to be higher than in skin, where an average of about was found in 8 excised heel skin samples,20 and an average of about in 28 excised abdominal tissue samples.21
High-Performance Liquid Chromatography
The RRS detection method is sensitive only to the vibrations of the carbon backbone common to all carotenoid species and not to the molecular end groups. Therefore, it measures the integral optical response (“composite score”) of all carotenoid species present in the measured tissue volume. In contrast, the biochemical gold standard detection technique of high-performance liquid chromatography (HPLC) is capable of detecting each individual carotenoid subspecies. To gain insight into the carotenoid composition of human bone and their relative abundance, we analyzed biopsied samples with the HPLC method as a next step.
To achieve high-detection accuracy (), relatively large bone samples () were used. Following biopsy and weighing, the collected samples were immediately frozen until later HPLC analysis.22 The protocol involved weighing the samples again, transferring them into tubes with 430-μl phosphate buffered saline (PBS) and 70-μL collagenase ( PBS), incubating them for 1 h at 37°C, and adding glass beads and a 2 mL mixture of 50% methanol/50% THF and 100 μL of tocol (internal standard). Further, the samples were mixed to macerate the tissue and to extract the carotenoids. Samples were saponified overnight at 4°C by the addition of 100-μL of 10% pyrogallol and 1 mL of 40% potassium hydroxide. Samples were extracted two times with 5-mL hexane and dried over sodium sulfate. The combined hexane extract was dried in a SpeedVac™. The sample for astaxanthin analysis was extracted with acetone rather than and was hydrolyzed using cholesterol esterase. It was separated by HPLC using a Diol column.
The HPLC system consisted of a computer data system, an autosampler for maintaining samples at 20°C, a column heater at 31°C, and a diode array detector (ThermoSeparation Products, Fremont, California). The separation was performed isocratically on a Spherisorb ODS2 column (3 μm, with titanium frits; ES Industries, West Berlin, New Jersey) protected by a Javelin™ guard column containing a similar stationary phase (Thermo Electron Corp., Bellefonte, Pennsylvania). The mobile phase consisted of acetonitrile/dioxane/isopropanol/triethylamine (80/15/5/0.1) at a flow rate of . The alcohol component contained 100-mM ammonium acetate. The diode array detector with light pipe flow cell was programmed to monitor tocol and phytoene at 290 nm, phytofluene at 325 nm, and carotenoids at 450 nm.
Linear calibration curves were prepared for three concentrations of analytes, which spanned the physiological levels of micronutrients in serum. The calibrants included are astaxanthin, lutein, zeaxanthin, -cryptoxanthin, -cryptoxanthin, lycopene, -carotene, -carotene, phytoene, and phytofluene.
A typical HPLC chromatogram of a biopsied bone sample is shown in Fig. 5, demonstrating good signal-to-noise ratios for all carotenoid peaks and allowing us to determine their individual concentrations in bone with high accuracy. Importantly, a wide spectrum of carotenoids in bone is apparent including lutein, zeaxanthin, - and -cryptoxanthins, trans- and cis-lycopene, -carotene, trans- and cis--carotene, phytoene, and phytofluene.
To further establish a correlation between optically and biochemically derived tissue carotenoid content, and in this way to validate a quantitative optical detection approach, we selected nine biopsied samples with low-, medium-, and high-carotenoid contents, respectively, as determined via RRS measurements, and subsequently determined their carotenoid content via HPLC analysis. The results are shown in Fig. 6, yielding a satisfactory high-correlation coefficient of . The blue-light excitation RRS measurements are not sensitive to phytoene and phytofluene, which due to their relatively short conjugation lengths absorb at much shorter wavelengths in the UV.15 In calculating the total carotenoid content by HPLC, their respective concentrations were subtracted.
The obtained correlation between optically and biochemically derived bone carotenoids is surprisingly high in view of the fact that bone tissue provides a rather heterogeneous light-tissue interaction scenario. Bone morphology and light propagation are conceptually illustrated in Fig. 7. It shows idealized cross-sections through extreme cases of healthy and osteoporotic spongy bone samples, respectively, on a millimeter-scale spatial resolution, with the laser light excitation area inside the bone tissue indicated as a shaded hemisphere. The light excitation encounters carotenoid-containing bone marrow located inside the spongy bone cavities as well as their carotenoid-free calcium walls. The wall thicknesses are known to vary between patients, thinning considerably in patients with osteoporosis, for example. As a consequence, the morphological bone differences between subjects will lead to differences in the spatial overlap of the light excitation and the carotenoid-containing soft tissue (marrow) mass located inside the cavities and influence the obtainable correlation between optically and biochemically determined bone carotenoid content.
All carotenoids found in human skin24 apparently exist also in human spongy bone. To illustrate this point more quantitatively, we show in Fig. 8 the relative abundances of carotenoids measured via HPLC analysis in nine excised bone and eight skin samples, respectively, with samples originating from different subjects. In bone, the concentrations of lutein, zeaxanthin, and -crytoxanthin appear to be similar to skin, while the concentrations of carotene, phytoene and phytofluene appear to be higher (up to a factor of 2). Potentially these tissue differences could be important, but a larger number of samples/subjects are needed for sufficient statistical power.
While RRS spectroscopy is well suited for the detection of carotenoids with high-molecular specificity and, in principle, can also be used in filter-based spatial imaging configurations, its implementation is rather complex, requiring relatively complicated 488-nm laser excitation with sequential detection on and off the Raman line.25 A potentially much simpler methodology for the remote optical detection of bone carotenoid status during total knee arthroplasty is reflection-based imaging. To develop this methodology for bone carotenoid detection, we first measured reflection spectra of bone and surrounding tissue in a spectrally resolved nonimaging configuration to determine if the characteristic yellow tissue colorations due to carotenoids can be quantified with sufficient sensitivity. As a setup, we used broadband white light illumination over the 380 to 750-nm spectral range in combination with a spectrograph/CCD array detection system, analyzing the light that is reflected back from the sample in 180-deg back-reflection geometry. Essentially, the setup is the same as previously used for the measurement of carotenoids in human skin.26 The apparent absorbance, , of the sample is calculated as for each spectral data point, where is the measured reflectivity.
In Fig. 9, reflection-derived apparent absorption spectra in the 380 to 750-nm spectral range are shown for excised spongy bone, fat, and cartilage samples. For the bone and fat samples, strong carotenoid vibronic absorption bands are clearly discernible, superimposed on a scattering background, at and 480 nm, with potentially interfering tissue chromophores largely absent. In cartilage, no carotenoids are detectable. The concentration of hemoglobin differs between tissues types, as can be seen from differences in its characteristic single-band absorption strength at and its double-band absorption structure at .
The apparent absorption spectra of spongy bone and fat samples exhibit nearly identical combined absorption and scattering behaviors in the red spectral region between 600 and 750 nm, as can be seen from Fig. 10. This spectral region can therefore be used as an effective background absorption/scattering level for the calculation of carotenoid levels from their absorption in the blue spectral region. Absorbance differences in the spectral range from to 520 nm would then correlate with different tissue carotenoid concentrations. Spectral regions suitable for quantitative reflection-based imaging of carotenoid levels in spongy bone and fat tissues are indicated in Fig. 10 as “carotenoid detection band” and “reference band,” respectively. The spectral location of the reference band is chosen to avoid potential absorption overlap from confounding chromophores, such as hemoglobin, and at the same time to provide a measure for the scattering background that is comparable with the scattering background under the “carotenoid detection band.”
To prove this concept, we constructed a filter-based reflection imaging setup, which is shown schematically in Fig. 11. Light from a spectrally broad “white” light emitting diode (LED) is routed to the surface of the tissue sample with the help of a simple lens projection configuration. The overall excitation light power of the LED was about 5 mW, corresponding to an excitation light intensity of . The light scattered back from the tissue is collected by an objective lens and transmitted sequentially through two band-pass filters with peak transmissions at 620 and 488 nm, respectively, mounted on a filter wheel. The bandwidths of the transmitted light were 10 nm in both cases. The spectrally filtered images were recorded with the array of a CCD camera (Santa Barbara Instrument Group, Santa Barbara, CA, Model ST-9; pixel size ), permitting digital image acquisition with 16 bits of grayscale. The camera was positioned about 40 cm from the object plane, thus viewing the samples with a 29-deg field-of-view.
For the generation of bone carotenoid reflectivity maps and corresponding optical densities, we first recorded a “red” tissue image along with an image of a reflection standard (“Spectralon,” Labsphere, Inc., North Sutton, NH) placed in the same object plane [see Fig. 12(a)]. Subsequently, we normalized each red tissue image pixel to the reflection standard and calculated the common logarithm according to
Subsequently, the two optical density maps can be subtracted to generate pixel line plots across directions of interest, such as through the center of the sample, as shown in Fig. 12(c) for a spongy bone sample exhibiting carotenoid levels as high as 0.5 optical density units on average over most of its central surface. Importantly, this reflection imaging-derived concentration is about the same as the concentration derived via direct absorption measurement of the extract, according to Fig. 4 (estimating an effective path length of for reflection in the blue wavelength region in the bone and taking into account the 1 cm path length in the extract).
Our results show that carotenoids exist in human bone and surrounding fatty tissue in high concentrations and in the same variety of subspecies found in skin and they are known to circulate in the blood stream. Carotenoid levels can be detected quantitatively in exposed bone and surrounding tissue in noncontact detection configurations using RRS. A proof of concept experiment shows that reflection spectroscopy could be a viable alternative. Its implementation is relatively simple, essentially requiring only spectrally selective, filter-based photography, and it can generate spatially resolved quantitative information on tissue carotenoid levels. The method would therefore be applicable in clinical settings where exposed tissues could be measured during surgery, such as in total knee arthroscopy.
Measuring or estimating the carotenoid levels of a patient’s bones or surrounding tissue provide many potential benefits.27 For example, the patient and doctor can be better guided in selecting an appropriate bone integration implant device (e.g., cemented implant devices may be more appropriate when bone carotenoid levels are low). Similarly, if a patient’s bone carotenoid content is known or estimated to be low, the patient can be encouraged to make dietary changes (e.g., consuming carotenoid-rich foods and/or carotenoid nutraceuticals) or to select medical regimens for improving bone health (e.g., combining prescription drugs with exercise regimens and nutritional changes). Further, a known or measured bone carotenoid level may be used to predict the health of other organs or systems (e.g., cardiovascular health, neurologic health, mental health, eye health, insulin sensitivity, etc.).
Since the carotenoid species found in bone are similar to skin, it may be possible to establish skin carotenoid measurements as a surrogate marker for these internal tissues. A clinical trial is under way to investigate a likely correlation between the carotenoid concentration of a patient’s exposed bone tissue with skin, which can be more easily assessed optically. A resulting correlation table could be particularly useful when a patient’s carotenoid concentrations are optically tracked over time.
Currently, in orthopedic surgery, there is much discussion and disagreement on the relative advisability and durability of knee, hip, and shoulder implants either as cemented by methyl methacrylate or press fit (cement-less) for direct bone integration.28 Press-fit knee implants achieving successful bone integration and bonding are often recommended for 50- to 70-year-old patients. However, many surgeons remain reluctant to use press-fit implants for fear of unsuccessful bone integration. This study looks at measuring the relative concentration of carotenoids exposed during routine bone cuts as a possible predictor of bone health and therefore a predictor of robust bone integration and bonding.