Near-Infrared Time-Resolved Spectroscopy for Assessing Brown Adipose Tissue Density in Humans: A Review

Brown adipose tissue (BAT) mediates adaptive thermogenesis upon food intake and cold exposure, thus potentially contributing to the prevention of lifestyle-related diseases. ¹⁸F-fluorodeoxyglucose (FDG)–positron emission tomography (PET) with computed tomography (CT) (¹⁸FDG–PET/CT) is a standard method for assessing BAT activity and volume in humans. ¹⁸FDG–PET/CT has several limitations, including high device cost and ionizing radiation and acute cold exposure necessary to maximally stimulate BAT activity. In contrast, near-infrared spectroscopy (NIRS) has been used for measuring changes in O₂-dependent light absorption in the tissue in a non-invasive manner, without using radiation. Among NIRS, time-resolved NIRS (NIR_TRS) can quantify the concentrations of oxygenated and deoxygenated hemoglobin ([oxy-Hb] and [deoxy-Hb], respectively) by emitting ultrashort (100 ps) light pulses and counts photons, which are scattered and absorbed in the tissue. The basis for assessing BAT density (BAT-d) using NIR_TRS is that the vascular density in the supraclavicular region, as estimated using Hb concentration, is higher in BAT than in white adipose tissue. In contrast, relatively low-cost continuous wavelength NIRS (NIR_CWS) is employed for measuring relative changes in oxygenation in tissues. In this review, we provide evidence for the validity of NIR_TRS and NIR_CWS in estimating human BAT characteristics. The indicators (Ind_NIRS) examined were [oxy-Hb]_sup, [deoxy-Hb]_sup, total hemoglobin [total-Hb]_sup, Hb O₂ saturation (StO_2sup), and reduced scattering coefficient (${\mu }_{\text{s sup}}^{\prime }$) in the supraclavicular region, as determined by NIR_TRS, and relative changes in corresponding parameters, as determined by NIR_CWS. The evidence comprises the relationships between the Ind_NIRS investigated and those determined by ¹⁸FDG–PET/CT; the correlation between the Ind_NIRS and cold-induced thermogenesis; the relationship of the Ind_NIRS to parameters measured by ¹⁸FDG–PET/CT, which responded to seasonal temperature fluctuations; the relationship of the Ind_NIRS and plasma lipid metabolites; the analogy of the Ind_NIRS to chronological and anthropometric data; and changes in the Ind_NIRS following thermogenic food supplementation. The [total-Hb]_sup and [oxy-Hb]_sup determined by NIR_TRS, but not parameters determined by NIR_CWS, exhibited significant correlations with cold-induced thermogenesis parameters and plasma androgens in men in winter or analogies to ¹⁸FDG–PET. We conclude that NIR_TRS can provide useful information for assessing BAT-d in a simple, rapid, non-invasive way, although further validation study is still needed.

Introduction

Human adipose tissues are of a variety of types, such as white (WAT) and brown adipose tissue (BAT) (1). WAT is capable of depositing extra-energy as triglyceride droplets under conditions where energy intake is greater than its expenditure. In contrast, BAT promotes non-shivering thermogenesis to respond to decreases in core body temperature and, in contrast to WAT, is characterized by an abundance of mitochondria and vasculature. BAT has been extensively investigated in animals, and it has been determined that BAT-specific uncoupling protein (UCP)-1, mainly stimulated upon β₃-adrenergic activation by cold and/or dietary intervention, enables BAT to dissipate free energy to heat by proton discharge through the inner mitochondrial membrane (2, 3). BAT has drawn renewed attention since 2009, with several papers being published that report the existence of BAT deposits in adult humans (4–7), which had previously been thought to be lost during the process of maturation. Human BAT is reported to be related to lower adiposity [body mass index (BMI), the percentage of whole body fat (%BF), and visceral fat area (VFA)] (6–9) and increased glucose sensitivity (10). In experimental studies, repeated exposure to cold environment enhanced the BAT activity and improved glucose tolerance in obese counterparts (11) and patients with type 2 diabetes mellitus (12) as well as in healthy individuals (9, 13, 14). Thus, increasing BAT activity or volume may aid in combatting obesity and chronic diseases, such as type 2 diabetes mellitus.

It is well-known in humans that BAT can be evaluated by ¹⁸F-fluorodeoxyglucose (FDG)–positron emission tomography (PET) with computed tomography (CT) (¹⁸FDG–PET/CT) under cold-stimulated environments (3, 4, 6, 15). However, ¹⁸FDG–PET/CT has several limitations, including the enormous cost of the device and ionizing radiation exposure, and acute cold exposure—necessary to maximally stimulate BAT activity (16), which make a longitudinal ¹⁸FDG–PET/CT study difficult and disrupt interventional research, specifically longitudinal ones in humans. Cold exposure is primarily required in ¹⁸FDG–PET/CT studies to activate human BAT, and various protocols have been applied in the past. While standardized guidelines have recently been proposed, differences between protocols remain a significant obstacle to the comparison of observations from different studies (17).

Other non-invasive technologies have been utilized for evaluating BAT characteristics in humans, such as magnetic resonance imaging (MRI) (18, 19), local skin thermal measurements (19), infrared thermography (20), and contrast ultrasound (21). A recent review on the detection of BAT using these non-invasive technologies can be found elsewhere (22). Regarding MRI technologies, proton-density fat fraction (PDFF) values are widely used to distinguish BAT from WAT (23, 24). However, the PDFF range in the supraclavicular region widely varies among individuals, which makes the differentiation of BAT from WAT difficult, although several new technologies, such as the measurement of T2^* relaxation and diffusion-weighted imaging are under investigation (22). The local skin thermal measurements have been used for monitoring cold-induced temperature changes in the supraclavicular skin with infrared thermography (25, 26). However, heat measurements could be influenced by the tissue conductive properties and thickness of the subcutaneous adipose tissue (27), and the individual vasomotor response (28); these evaluation obstacles should be solved in the future.

In addition, near-infrared spectroscopy (NIRS) is a relatively newly introduced methodology to monitor BAT properties (29). The basis for the application of NIRS to evaluate BAT properties is that the microvascular bed, as evaluated by total hemoglobin (Hb) concentration [total-Hb]_sup in the supraclavicular region, is more abundant in BAT than in WAT (30). Furthermore, NIR time-resolved spectroscopy (NIR_TRS) may be used to assess the density of the microvasculature as well as mitochondrial content in BAT by measuring the reduced scattering coefficient (${\mu }_{\text{s}}^{\prime }$), which reflects the in vitro mitochondrial content (31). BAT is a highly innervated tissue and is also highly perfused when exposed to cold (32). As the concentrations of oxygenated and deoxygenated Hb in the supraclavicular region ([oxy-Hb]_sup and [deoxy-Hb]_sup, respectively) are likely to change (especially [total-Hb]_sup, which reflects blood volume), it could be a valid measure of BAT vasculature.

The purpose of this article is to provide evidence concerning the ability of NIRS to evaluate BAT characteristics in humans. In this review, we included studies examining BAT characteristics using NIRS in humans: most studies used NIR_TRS (29, 33–39), a technology to quantify both absolute tissue absorption and scattering characteristics, while some utilized NIR continuous wave spectroscopy (NIR_CWS), an inexpensive technology that only provides relative values of tissue oxygenation (32, 40). First, we present how NIRS functions to evaluate tissue oxygenation and blood volume. Then, we provide data indicating whether BAT characteristics can be evaluated using NIR_CWS. The main body of the paper presents a series of evidence for NIR_TRS to assess BAT characteristics. The evidence tested comprises (1) the relationship between parameters determined using NIRS and those measured by ¹⁸FDG–PET/CT, (2) correlations between the NIRS parameters and cold-induced thermogenesis (CIT), (3) correspondence of the NIRS parameters to those reported using ¹⁸FDG–PET/CT regarding chronological and anthropometric data, (4) the correspondence between NIRS parameters and those reported with ¹⁸FDG–PET/CT in response to ambient temperature fluctuations, (5) the relationship between parameters determined using NIRS and plasma lipid metabolites, and (6) changes in NIRS parameters induced by supplementation with evidence-based thermogenic functional ingredients.

How NIRS Functions as Evaluating Tissue Oxygenation and Blood Volume

NIRS provides non-invasive monitoring of tissue oxygen and Hb dynamics in vivo (41–45). NIRS is able to monitor changes in O₂-dependent light absorption in the heme in the red blood cells circulating in biological tissues (46). There are mainly three types of NIRS devices: NIR_CWS, NIR_TRS, phase modulation NIR spectroscopy (NIR_PMS), etc. (46–48). The most popular NIRS devices use NIR_CWS that outputs only the qualitative tissue oxygenation. To calculate the changes in [oxy-Hb], [deoxy-Hb], [total-Hb], and Hb O₂ saturation (StO₂) using NIR_CWS, a combination of multiple-wavelengths can be adopted in accordance with the Beer–Lambert law. The main reason why quantitative data cannot be provided as continuous NIR light path traveled through tissues is unknown (42–45). However, spatially resolved NIRS (a type of NIR_CWS) is able to provide quantitative values considering several assumptions, although it is still unable to provide the tissue absorption and scattering properties.

On the other hand, NIR_TRS and NIR_PMS are more accurate, as they can quantify both tissue absorption and scattering characteristics. NIR_TRS emits ultrafast (100 ps) light pulses from the skin surface and measures the photon distributions across the biological tissue with a 2- to 4-cm distance from the light emission. NIR_TRS is able to quantitatively measure the absorption coefficient (μ_a), ${\mu }_{\text{s}}^{\prime }$, and then calculates light path length, tissue [oxy-Hb], [deoxy-Hb], [total-Hb], and StO₂ (44, 45, 48). The validity of the signal obtained by NIR_CWS and NIR_TRS has been confirmed in an in vitro experiment using highly scattering Intralipid™ (43, 44). Using this system, μ_a in the NIR range was found to be strongly correlated with [total-Hb] (43, 48). Furthermore, the study found a significant relationship between ${\mu }_{\text{s}}^{\prime }$ at 780 nm and the homogenized tissue mitochondrial concentration (31).

Studies Using NIR_CWS

Prior to the NIR_TRS study on human BAT, one study attempted to correlate oxygen dynamics using NIR_CWS and BAT parameters (32). In this cross-sectional study, adult human subjects (25 subjects; 15 women and 10 men; mean age ± SD, 30 ± 7 years) were assigned into high- (BMI, 22.1 ± 3.1) and low-BAT groups (BMI, 24.7 ± 3.9) based on the levels of ¹⁸F-FDG uptake in the cervical–supraclavicular region. It employed triple-oxygen PET scans (${\text{H}}_2^{15}$O, C¹⁵O, and ¹⁵O₂) and daily energy expenditure measurements under resting and mild cold (15.5°C) room conditions for 60 min using indirect calorimetry (32). They used a NIR_CWS parameter, adjusted supraclavicular StO₂ (adjStO₂), a balance between oxygen supply and uptake. In the high-BAT group, there was a significant negative correlation between oxygen consumption determined by PET scans and adjStO₂ (p = 0.02, r² = 0.46) in the supraclavicular region at rest and after the exposure to cold, indicating increased oxygen uptake in highly active BAT (32). However, it detected a limited effect on the difference in adjStO₂ between the two groups (32). It should be noted that the study presented several limitations to consider when interpreting its results: (1) a non-individualized cooling protocol was used; (2) only one NIR_CWS parameter, adjStO₂, was used in the analysis; and (3) no kinetics data determined by the NIR_CWS were provided.

Recently, in young healthy women, a study using the standardized cold exposure aimed to investigate the association between NIR_CWS parameters in the supraclavicular and forearm regions and BAT capacity assessed by ¹⁸FDG–PET/CT (40). Briefly, the subjects arrived at the laboratory (a room temperature of 19.5–20°C) and wore a temperature-controlled water circulation cooling vest for 60 min, and the individual temperature to be exposed was determined, namely at ~4°C above the threshold of shivering, 48–72 h prior to the ¹⁸FDG–PET/CT measurements. No association was found between any NIR_CWS indicators and maximal standardized uptake value (SUV_max) and mean standardized uptake value (SUV_mean) of the radioactivity both under thermoneutral and cold conditions. Thus, NIR_CWS would not be an appropriate technology to evaluate BAT capacity in this demographic. The lack of significant association between NIR_CWS parameters is mainly due to differences in the instrumentation to that used in NIR_TRS, which provides absolute values for tissue hemodynamics. Furthermore, NIR_CWS permits an ~15 mm depth of light penetration at a 30 mm input-output setups (44). However, the mean photon penetration would be deeper (~20 mm at the 30-mm input–output setups) and wider when NIR_TRS is used (49), which influences the differences in sensitivity between NIR_CWS and NIR_TRS with respect to BAT detection. Table 1 shows the relationship between [oxy-Hb]_sup, [deoxy-Hb]_sup, [total-Hb]_sup, StO_2sup, and adjStO_2sup determined by NIR_CWS and ¹⁸FDG–PET/CT parameters (SUV_max and SUV_mean), which have been documented in previous studies (32, 40). The only NIR_CWS parameter found to be significantly correlated with a ¹⁸FDG–PET/CT parameter, cold-induced oxygen uptake by BAT, is adjStO₂, and only in the high-BAT group.

TABLE 1

Table 1. Parameters obtained by continuous-wave near-infrared spectroscopy (NIR_CWS) for evaluating brown adipose tissue characteristics in the supraclavicular and control muscle regions.

Taken together, the studies using NIR_CWS (32, 40) present potential limitations beyond the fact that it cannot evaluate the absorption and scattering properties of the tissue, including that it is more sensitive to changes in the skin blood flow than NIR_TRS (32, 40). Therefore, NIR_CWS does not seem to be a valid measure for BAT function although emphasis should be placed in the need for further research examining this type of NIRS.

Studies Using NIR_TRS

Correlation Between Parameters Determined by NIR_TRS and ¹⁸FDG–PET/CT Parameters

It is speculated that, as BAT exhibits higher microvascular density and mitochondrial contents compared to WAT, NIR_TRS can be used for assessing the density of microvascular or mitochondrial content in BAT (BAT-d) by measuring [total-Hb]_sup and ${\mu }_{\text{s}}^{\prime }$ in the supraclavicular region (${\mu }_{\text{s sup}}^{\prime }$), which reflects the in vitro mitochondrial content (31). It may be expected that BAT would exhibit higher values for [total-Hb]_sup and ${\mu }_{\text{s sup}}^{\prime }$ than those exhibited by WAT. As NIR_TRS measures the average tissue hemoglobin concentration in a volume of 4 cm³ with a 3-cm optode separation (50), the ¹⁸FDG–PET/CT-determined SUV_mean is the most appropriate indicators for comparison to those determined using NIR_TRS. Although the use of other radiotracer techniques, such as triple-oxygen scans (${\text{H}}_2^{15}$O, C¹⁵O, and ¹⁵O₂) would be more appropriate to reflect BAT activity, ¹⁸FDG–PET/CT has been mostly used for determination of BAT activity owing to routine availability in a clinical science setup. First, 18 healthy male subjects (20.3 ± 1.8 years and BMI of 23.9 ± 3.1 kg/m²) were recruited to examine changes in NIR_TRS parameters under acute cold environment. The [oxy-Hb]_sup, [deoxy-Hb]_sup, [total-Hb]_sup, StO_2sup, and ${\mu }_{\text{s sup}}^{\prime }$ were compared between a room temperature under thermoneutral conditions and cold conditions (19°C) for 2 h. As there was no change in the [total-Hb]_sup and ${\mu }_{\text{s sup}}^{\prime }$ at 19°C compared to baseline conditions measured at 27°C, NIR_TRS can be used without the necessity of cold exposure (29). Second, to test this hypothesis, [total-Hb]_sup and ${\mu }_{\text{s sup}}^{\prime }$ were compared to the SUV_mean assessed by ¹⁸FDG–PET/CT (29) in a cross-sectional design. Twenty-nine healthy male subjects (23.3 ± 2.2 years and BMI of 21.6 ± 1.8 kg/m²) were recruited to evaluate the relationship between SUV_mean and NIR_TRS parameters. The results demonstrated that [total-Hb]_sup and ${\mu }_{\text{s sup}}^{\prime }$ under warm environment is significantly correlated with the SUV_mean under cold environment but only in the supraclavicular fossa, a region of BAT located (29). Other parameters, except adjStO₂ specifically in the supraclavicular region, also showed significant correlation with SUV_max and SUV_mean under cold environment (Table 2). Collectively, the [total-Hb]_sup, [oxy-Hb]_sup, and [deoxy-Hb]_sup show significant correlations to BAT activity determined by ¹⁸FDG–PET/CT. StO_2sup, however, proved to be inferior, and adjStO₂ was completely insensitive to changes in BAT activity (Table 2).

TABLE 2

Table 2. Parameters for evaluating brown adipose tissue characteristics using near-infrared time-resolved spectroscopy (NIR_TRS) in the supraclavicular and control muscle regions.

A 2-h cold exposure doubles the BAT blood flow (32), which appeared to be inconsistent with our observations. In an attempt to interpret this apparent discrepancy, we speculated that NIR_TRS parameters are susceptible to the change in the volume but less sensitive to the change in the flow. The blood flow can be calculated by multiplying the blood flow velocity by the cross-sectional area of the vessel (the volume). There is presently a lack of NIR_TRS-derived data concerning blood flow in BAT. Alternatively, while muscle blood flow increases by some 10-fold during peak exercise (51, 52), [total-Hb], an indicator of blood volume, monitored by NIR_TRS elevates only 1.1-fold (43). Thus, the change in blood volume measurable by NIR_TRS is marginal compared to increases in blood flow velocity during metabolic activation. Collectively, both ${\mu }_{\text{s sup}}^{\prime }$ and [total-Hb]_sup were evaluated using the NIR_TRS technique can be applied to assess BAT-d in humans and are equivalent to the active BAT intensity or the BAT volume, as measured by ¹⁸FDG–PET/CT under cold environment (29). Usually, to assign participants into high-BAT (BAT [+]) and low-BAT (BAT [–]) groups, a cutoff value of 2.0 for SUV_mean is applied. The accuracy of [total-Hb]_sup or ${\mu }_{\text{s sup}}^{\prime }$ in representing BAT activity was analyzed. Accordingly, the area under the receiver operating characteristic (ROC) curve was determined by SUV_mean of 2.0 nearest to (0, 1) for ${\mu }_{\text{s sup}}^{\prime }$ and [total-Hb]_sup (29). When 74.0 μM or 6.8 cm⁻¹ was selected as the cutoff value, meaning that [total-Hb]_sup or ${\mu }_{\text{s sup}}^{\prime }$ larger than 74.0 μM or 6.8 cm⁻¹, respectively, are regarded as BAT [+], ROC analysis yields results that are very good when compared to SUV_mean (29).

Correlation Between Parameters Determined by NIR_TRS and CIT

It is well-documented in rodents that the upregulation of the UCP-1 in brown adipocytes upon cold increases whole body oxygen consumption, termed as CIT. Although several authors have shown that CIT does not always reflect BAT activity (53), that the contribution of BAT thermogenesis to CIT is marginal (~10 kcal/day when maximally activated) (54), and no correlation is found between BAT and CIT (55), the magnitude of CIT is related to the amount of BAT activity and/or volume (9, 56–59). Thus, having already observed a significant correlation between NIR_TRS parameters and SUV_mean assessed by ¹⁸FDG–PET/CT (29) in humans, the validity of NIR_TRS parameters were further examined by comparing [total-Hb]_sup and ${\mu }_{\text{s sup}}^{\prime }$ to CIT in healthy individuals [age of 20.0 (median), 19.0 (the first quartile), and 21.0 (the third quartile) year; BMI of 24.2 (21.6, 25.7) kg/m²] with [total-Hb]_sup of 50–125 μM in a cross-sectional study (37). The participants sat for 20 min at 27°C with a light clothing, and NIR_TRS measurements were conducted for 5 min after fasting for 6–12 h. Then, the participants were tested at room temperature of 19°C for 2 h with their feet intermittently placed on a cloth-wrapped ice for ~4 min every 5 min (9). A significant correlation was found between [total-Hb]_sup, [oxy-Hb]_sup, or [deoxy-Hb]_sup only under thermoneutral conditions and CIT, but not between adjStO₂ or ${\mu }_{\text{s sup}}^{\prime }$ and CIT (37). In contrast, previous studies reported a significant correlation in the supraclavicular region between the adjStO₂ and oxygen consumption by BAT under cold environment and between ${\mu }_{\text{s sup}}^{\prime }$ and SUV_max and SUV_mean (29, 32). It is noted that a personalized cooling protocol may be a better procedure to induce a CIT response personalized to each individual (60). Collectively, although the [total-Hb]_sup, [oxy-Hb]_sup, and [deoxy-Hb]_sup are markers for BAT activity as evaluated by CIT, the adjStO₂ and ${\mu }_{\text{s sup}}^{\prime }$ become less sensitive to CIT (Table 3).

TABLE 3

Table 3. Relationship between parameters determined by near-infrared time-resolved spectroscopy (NIR_TRS) under thermoneutral or cold condition and pulmonary oxygen uptake during cold exposure.

Relationship Between NIR_TRS Parameters in the Supraclavicular Region and Chronological and Anthropometric Data

¹⁸FDG–PET/CT studies have revealed that a significant relationship exists between BAT activity and chronological and anthropometrical parameters (4, 5, 29, 56). Cold-stimulated ¹⁸FDG–PET/CT studies have shown that BAT activity negatively associated with age, sex, BMI, %BF mass, and VFA, and also that BAT was a significant independent determinant of glucose and HbA1c levels, after adjustment for age, sex, and body adiposity (10, 56).

A cross-sectional study using NIR_TRS demonstrated that [total-Hb]_sup under warm environment was negatively associated with age and body adiposity in 413 Japanese individuals [a median age of 43.0 (33.0–58.0, interquartile range) years, BMI of 22.5 (20.7–24.5) kg/m², and %BF of 26.8% (20.6–32.3%)] in winter (33) (Figure 1). With the exception of participants in their 20s, there were no sex-related differences in [total-Hb]_sup among the groups tested (Figure 1). Multivariate analyses revealed that the %BF and VFA were significantly negatively correlated with [total-Hb]_sup (33). The observation of the study was analogous to data acquired using ¹⁸FDG–PET/CT, indicating the usefulness of the parameter [total-Hb]_sup. In contrast, ${\mu }_{\text{s}}^{\prime }$ was found to be significantly negatively correlated with some of the anthropometrical parameters. Together, the [oxy-Hb] _sup and [deoxy-Hb] _sup displayed similar accuracy to the [total-Hb]_sup for detecting relationships with chronological and anthropometric data (Table 4).

FIGURE 1

Figure 1. Chronological and sex differences in terms of the concentration of the supraclavicular total hemoglobin [total-Hb], an indicator of brown adipose tissue (BAT). The supraclavicular [total-Hb] potentially containing BAT. The BAT-positive rate (SUV_max > 2.0) is indicated in the bottom of the figure based on previous studies (29, 33). The values are presented as means ± standard error (SE), adjusting for body mass index, body fat ratio, and visceral fat area. ^©SPIE. Reproduced by permission of the publisher. Adopted from reference (33).

TABLE 4

Table 4. Relationship between parameters in the supraclavicular region obtained by near-infrared time-resolved spectroscopy (NIR_TRS) and anthropometric and body composition parameters.

Changes in NIR_TRS Parameters in the Supraclavicular Region in Response to Ambient Temperature Fluctuations

BAT increases in winter according to ¹⁸FDG–PET/CT studies (4, 56–59). However, one study reported that early winter showed higher BAT activity than late winter or early spring (61). A cross-sectional study (35) reported that [total-Hb]_sup was higher in winter than in summer. It has also been reported that a lower average ambient temperature during the 4–6 weeks before the measurement day increases [total-Hb]_sup (33). This finding is consistent with previous findings reporting that, while BAT activity rose during the winter, a few months are needed for the increase in BAT activity after a decrease in the air temperature (58). A longitudinal study using the same healthy subjects (men/women, 35/23; mean age, 37.4 years; mean BMI, 22.5 kg/m²; BAT positive rate, 48%) in summer and winter under thermoneutral conditions revealed a significant increase in [total-Hb]_sup, but not in the reference region or in the ${\mu }_{\text{s}}^{\prime }$ from any regions (37). It is unclear why ${\mu }_{\text{s sup}}^{\prime }$ region did not change between summer and winter. However, it is speculated that if triglyceride droplet in the supraclavicular area decreases in winter owing to the increase in BAT (or beiging), the ${\mu }_{\text{s}}^{\prime }$ should decrease because WAT (triglyceride droplet) obtains high scattering characteristics (62). Thus, even increasing the mitochondria content (the increase in ${\mu }_{\text{s}}^{\prime }$) would offset the decrease in WAT (the decrease in ${\mu }_{\text{s}}^{\prime }$), indicating that [total-Hb]_sup may be a better indicator of BAT activity than ${\mu }_{\text{s sup}}^{\prime }$. We demonstrated seasonal changes in other NIR_TRS parameters, which supplement previously published findings in Table 5 (35, 37). The [oxy-Hb]_sup and the [total-Hb]_sup obtain similar tendency for monitoring seasonal fluctuations in BAT-d. In winter, the decrease in the deltoid [deoxy-Hb] demonstrated that muscle metabolism blunted in winter and that this is less reliable than [oxy-Hb]_sup or [total-Hb]_sup (Table 5).

TABLE 5

Table 5. Changes in parameters obtained by near-infrared time-resolved spectroscopy (NIR_TRS) by seasonal temperature changes.

Collectively, the [oxy-Hb]_sup, [total-Hb]_sup, StO_2sup, and adjStO₂ can detect seasonal fluctuations of BAT-d, which is consistent with the findings of previous ¹⁸FDG–PET/CT studies (4, 56–59).

Correlation Between NIRS Parameters in the Supraclavicular Region and Lipid Metabolites

Finding blood biomolecules correlated with BAT characteristics would permit us to advance human BAT studies because PET/CT studies may be difficult owing to ionizing radiation and cold exposures. Studies on lipidomic profiles have clarified BAT and WAT characteristics according to muscle contractions or cold environment (11, 12, 14, 16). BAT characteristics are related to unique profiles of lipid metabolites, such as the concentration of lysophosphatidylcholine-acyl (LysoPC-acyl) C16:0 in humans (63), and the concentration of phosphatidylethanolamine (PE) in the BAT and WAT was decreased in high-fat diet-fed mice (14). The relationships have been examined in the winter and summer between [total-Hb]_sup, a parameter for evaluating BAT-d, measured using NIR_TRS and plasma lipids in humans (38). Healthy volunteers with [total-Hb]_sup values over 74.0 μM (high BAT-d) were studied (n = 23) and control volunteers with lower [total-Hb]_sup values <70.0 μM (low BAT-d) (n = 23) were tested. Ninety-two plasma samples were examined (23 men and 23 women, aged 21–55; BMI, 21.9 ± 3.0 kg/m², %BF 23.3 ± 8.0%), in summer and winter. Using liquid chromatography-time-of-flight-mass spectrometry, plasma lipid profiles were determined. The [total-Hb]_sup was determined as a parameter of BAT-d using NIR_TRS under thermoneutral conditions. Body composition parameters, such as %BF and VFA, were examined. Univariate and multivariate regression analyses were used to determine factors affecting [total-Hb]_sup. In men, there were 37 metabolites showing positive correlations and 20 metabolites showing negative correlations (P < 0.05) with [total-Hb]_sup, respectively. After the Q values were obtained by correcting false discovery rate, only androgens (testosterone, androstanedione, dehydroandrosterone, dehydroepiandrosterone, or epitestosterone) showed a significant (Q < 0.05) positive correlation with [total-Hb]_sup in men in winter. Multivariate regression analysis revealed that [total-Hb]_sup showed a significant correlation with androgens in men and VFA in women in winter. Notably, the [total-Hb]_sup showed a significant relationship with androgens in winter in men but did not with any body-composition characteristics, such as whole body and visceral adiposity, which are generally associated with [total-Hb]_sup. Although androgens deteriorated the BAT capacity in vitro (64), testosterone induced a preferable effect on BAT activity, body adiposity, and energy expenditure in animal models (65–67). Thus, BAT characteristics might be predicted by measuring plasma androgens as a biomarker in men in the winter. However, further detailed research is needed to discover biomarkers that predict BAT in women.

Changes in NIRS Parameters in the Supraclavicular Region by Thermogenic Functional Ingredients

Recent studies have demonstrated that BAT can improve health status and has a protective effect on lifestyle-related diseases (4–11, 13, 14). Consequently, research has been focused on finding methods for effectively enhancing BAT activity and/or mass (9, 11–14). Developed strategies include cold acclimation (9, 11–14) and acute treatment of β₃-adrenergic receptor (AR) agonists in humans (68). However, cold exposure intervention would not be easy to apply to daily life (9), and β3-AR agonists may elicit unpreferable influence, including a risk for hypertension and increased susceptibility to arterial sclerosis (68). Recent investigations have revealed the mechanisms underlying the effects of thermogenic food ingredients. Pathways involved include the transient receptor potential channels (TRP)-BAT axis, a site of adaptive thermogenesis evoked by β-adrenoceptor activation (69). The TRP-BAT axis comprises the activation of cold-sensitive TRP channels located in peripheral tissues, such as the skin and intestines. The activation of TRP channels results in the signal delivery through the afferent nerve to the hypothalamus, which then evokes sympathetic nerve activation within BAT. This causes norepinephrine (NE) release, initiating β-adrenergic tracts to brown adipocytes and eliciting UCP1 upregulation and adaptive thermogenesis (69). In contrast to cold exposure intervention, functional food ingredients may be easily incorporated into daily life. This has been confirmed in animals and humans and includes capsinoids as TRP vanilloid 1 agonists, catechins as TRPA1/V1 agonists, and so on (70). Furthermore, they have the benefit of having no apparent side effects (9, 34, 36, 69, 70).

Among thermogenic food ingredients, substances, such as capsiate are known to increase BAT activity (9, 70). Previously, the effect of capsiate on [total-Hb]_sup, determined by the NIR_TRS, was examined (34). Twenty healthy individuals [capsiate group (n = 10) vs. placebo group (n = 10), 20.7 ± 1.2 years vs. 20.9 ± 0.9 years; BMI, 21.4 ± 1.8 vs. 21.9 ± 1.0 kg/m²; %BF, 21.3 ± 7.6% vs. 22.9 ± 8.7%] were supplemented either with capsiate (9 mg/day) daily for 8 weeks or a placebo in a paralleled, double-blind manner, and [total-Hb]_sup was measured during the treatment period, and for an 8-weeks follow-up period under thermoneutral conditions (34). The study also measured BAT activity with ¹⁸FDG–PET/CT under cold-exposure conditions as previously reported (29). This was only done twice (not every 2 weeks), pre- and post-supplementation, to reduce participant exposure to ionizing radiation. The study demonstrated a parallel change in BAT-d (+46.4%, P < 0.05) pre- and post-supplementation, evaluated as [total-Hb]_sup, or as BAT activity (+48.8%, P < 0.05) evaluated as the SUV_max, a parameter of the BAT capacity, by ¹⁸FDG–PET/CT, after the supplementation of thermogenic capsiate (Figures 2, 3). During the 8-weeks follow-up period, the [total-Hb]_sup decreased both in the capsiate and placebo groups; the decrease was greater in the capsiate group (albeit not significantly, P = 0.07) compared to that of the placebo group.

FIGURE 2

Figure 2. (A) Typical images of the uptake of ¹⁸F-fluorodeoxy glucose (FDG) (mean standardized uptake value) before and after the 6-weeks capsiate supplementation in the supraclavicular region. (B) Maximal standardized uptake value (SUV_max) pre- and post-supplementation. ^©SPIE. Reproduced by permission of the publisher. Adopted from reference (34).

FIGURE 3

Figure 3. The concentration of total hemoglobin [total-Hb], an indicator of brown adipose tissue (BAT) in (A) the supraclavicular fossa potentially containing BAT and (B) the deltoid, a control region. Adopted from Nirengi et al. (34). ^©SPIE. Reproduced by permission of the publisher.

Previous studies examined whether a catechins-rich green tea extract increases energy consumption in humans (71–74). Animal studies have shown that catechin intake increases BAT, the effects of which were abolished when the β-blocker was administrated (75, 76). Thus, we used NIR_TRS under thermoneutral conditions to test the effect of sustained catechin-rich ingredient (540 mg/day) intake on [total-Hb]_sup and investigated potential associations between changes in [total-Hb]_sup and body adiposity in 22 healthy women college students [catechin group (n = 10) vs. placebo group (n = 11), 21.1 ± 2.0 years vs. 20.5 ± 2.1 years; BMI, 21.1 ± 1.3 vs. 20.9 ± 1.6 kg/m²; %BF, 24.0 ± 3.5% vs. 25.8 ± 7.6%] (36). That study revealed that the [total-Hb]_sup was elevated by 19% in the catechin group only after 12 weeks (36). As for the ${\mu }_{\text{s}}^{\prime }$, which was not documented in the previous study, it did not change during catechin ingestion. There was a significant negative relationship between the enhancement in [total-Hb]_sup and the decrease in extramyocellular lipids, an indicator for possible insulin insensitivity (77), in the vastus lateralis muscle determined by proton-magnetic resonance spectroscopy (r = −0.66, P < 0.05).

After further analysis, some of which has not been documented in the previous study (34), capsiate supplementation was shown to cause a significant increase in [total-Hb]_sup, [oxy-Hb]_sup, and [deoxy-Hb]_sup and, upon its withdrawal, a decrease in [total-Hb]_sup, [oxy-Hb]_sup, StO_2sup, and adjStO₂ (Table 6). Similarly, by the catechins intervention, the [oxy-Hb]_sup, [deoxy-Hb]_sup, StO_2sup, and adjStO₂ obtain the same result as the [total-Hb]_sup for assessing increases in BAT-d (36). Collectively, studies into functional ingredient supplementation using NIR_TRS suggest that [oxy-Hb]_sup and [total-Hb]_sup are particularly suitable for the evaluation of BAT-d in intervention studies where the use of ¹⁸FDG–PET/CT is not applicable (Table 6).

TABLE 6

Table 6. Changes in parameters obtained by near-infrared time-resolved spectroscopy (NIR_TRS) for evaluating changes in brown adipose tissue characteristics by the supplementation with thermogenic ingredients.

Limitations and Perspectives

The studies using NIRS contain several limitations. Several optical issues should be considered, as the multilayer, inhomogeneous tissue property created by skin, adipose tissue, and muscle may affect in vivo tissue scattering and absorption characteristics and modulation of optical path. In a study (39), the optical characteristics in the deltoid, abdominal, and supraclavicular regions were tested using NIR_TRS. The results indicate that there are unique region-specific relationships between [total-Hb] and ${\mu }_{\text{s}}^{\prime }$, suggesting that examining the [total-Hb]–${\mu }_{\text{s}}^{\prime }$ relationship is a practical way to distinguish BAT from other tissues. It could be noted that due to the nature of optical measurements, the placement of the optodes for the NIR_TRS must be always secure and in the same area, especially during longitudinal studies. Although NIR_TRS is able to quantify tissue oxygen dynamics, the values are affected by optical characteristics underlying subcutaneous adipose tissue in the supraclavicular region, which varies depending on the body composition of subjects, thereby influencing NIR_TRS measurements. The reason is that the values obtained is diluted by the lower [Hb] in the subcutaneous adipose tissue (78). The [total-Hb]_sup values can be recalculated by considering the thickness of the adipose layer (79).

As no change in the [total-Hb]_sup and ${\mu }_{\text{s sup}}^{\prime }$ was observed during 2-h conditions at 19°C compared to baseline conditions at 27°C (29), NIR_TRS cannot detect changes in BAT characteristics responding to an acute cold exposure in nature because NIR_TRS is insensitive to changes in the blood flow (33, 35–37). However, a newly developed NIR_TRS system contains six wavelengths (760, 800, 830, 908, 936, and 976 nm), of which the latter three wavelengths are adopted to detect optical characteristics of lipids and water (80). This system could provide information on the changes in tissue water and lipid content in response to acute interventions, such as experimental cold exposure, which cannot be obtained using the conventional three-wavelength NIR_TRS system. The new six-wavelength NIR_TRS system could contribute further insight on the chronic as well as acute responsiveness of BAT metabolism in humans.

Finally, future studies should obtain further evidence to validate BAT evaluation using NIR_TRS because¹⁸FDG–PET/CT measurements include several limitations. BAT mainly consumes intracellular lipids, as well as plasma non-esterified fatty acids and those derived from lipoproteins—whereas ¹⁸FDG–PET/CT measures a glucose analog. The lack of standardization when quantifying BAT by ¹⁸FDG–PET/CT is also a problem. Thus, additional experiments to reach this conclusion are required, such as (1) examining whether NIR_TRS parameters actually represent the in vivo mitochondrial density of BAT, or are related to molecules implicated in the vascularization and thermogenesis of BAT [e.g., vascular endothelial-cell growth factor (VEGF), UCP-1, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α)]. This could be carried out by taking human biopsies from the supraclavicular area and examining the relationship between NIR_TRS parameters and the molecular signature of this tissue; (2) using other radiotracers beyond ¹⁸F-FDG, such as ¹⁵O, ${\text{H}}_2^{15}$O, C¹⁵O, or ¹¹C-acetate, which will allow to measure the real oxygen consumption, tissue perfusion, and metabolic activity of human BAT and which are more likely to represent the thermogenic nature or activity of this tissue than ¹⁸F-FDG; (3) carrying out studies where the kinetics of NIR_TRS are related to the kinetics of the metabolic activity of BAT (dynamic PET/CT); (4) using different cooling protocols, aiming to standardize the cooling stress to which individuals are submitted (avoiding potential biases in individual BAT activation); and (5) performing reliability studies to examine whether NIR_TRS measures can be replicated in the short and long term.

Conclusion

Correlation coefficients are presented for parameters determined by NIRS and ¹⁸FDG–PET/CT, CIT, or anthropometric and body composition parameters. Significant correlations were found between [total-Hb]_sup, [oxy-Hb]_sup, [deoxy-Hb]_sup, ${\mu }_{\text{s sup}}^{\prime }$, StO_2sup, or adjStO₂ and ¹⁸FDG–PET/CT indicators; between [total-Hb]_sup, [oxy-Hb]_sup, or [deoxy-Hb]_sup and CIT; and between [total-Hb]_sup, [oxy-Hb]_sup, or [deoxy-Hb]_sup and anthropometric and body composition indicators. The percentage changes in NIR_TRS parameters as a consequence of either seasonal temperature fluctuations or dietary supplementation with thermogenic ingredients are presented. Seasonal temperature fluctuations influenced [total-Hb]_sup, [oxy-Hb]_sup, StO_2sup, and adjStO₂. Studies on thermogenic capsinoid or catechin supplementation revealed a significant increase in [total-Hb]_sup, [oxy-Hb]_sup, and [deoxy-Hb]_sup. Upon withdrawal of these supplements, a decrease in [total-Hb]_sup, [oxy-Hb]_sup, StO_2sup, and adjStO₂ was seen. Recently, androgens were found to show a significant positive correlation with [total-Hb]_sup only in men in winter. Thus, BAT characteristics might be predicted by measuring plasma androgens as a biomarker in men in the winter.

We conclude that NIR_TRS would be a useful non-invasive technology for assessing BAT-d, although further validation is still needed. Among the parameters evaluated by NIR_TRS, the [oxy-Hb]_sup as well as [total-Hb]_sup would be applicable to assessing BAT characteristics in both cross-sectional and interventional studies.

Author Contributions

TH, SN, SF, and YK collected the relevant literature and wrote the manuscript. SA, RK, MK, and TE assisted in illustrations, formatting, and collection of literature. NS, MM, MS, and TY coordinated and edited the relevant discussion on PET/CT measurements and BAT.

Funding

We have been granted by Japan Society for the Promotion of Science (15H03100 and 19H04061), which is an independent administrative institution, established by way of a national law for the purpose of contributing to the advancement of science in all fields of the natural and social sciences and the humanities.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer FA declared a past co-authorship with one of the authors TH to the handling editor.

Acknowledgments

This work was supported by JSPS KAKENHI (15H03100 and 19H04061).

Abbreviations

adjStO₂, adjusted supraclavicular StO₂; AR, β3-adrenergic receptor; BAT, brown adipose tissue; BAT-d, vascular or mitochondrial density in BAT; BMI, body mass index; CIT, cold-induced thermogenesis; CT, computed tomography; deoxy-Hb, deoxygenated Hb; FDG, ¹⁸F-fluorodeoxyglucose; Hb, hemoglobin; NE, norepinephrine; NIRS, near-infrared spectroscopy; NIR_CWS, NIR continuous-wave spectroscopy; NIR_TRS, NIR time-resolved spectroscopy; oxy-Hb, oxygenated Hb; StO₂, Hb O₂ saturation; PET, positron emission tomography; ROC, receiver operating characteristic; total-Hb, total hemoglobin; TRP, transient receptor potential channels; SUV_max, maximal standardized uptake value; SUV_mean, mean standardized uptake value; WAT, white adipose tissue; μ_a, absorption coefficient; ${\mu }_{\text{s}}^{\prime }$, reduced scattering coefficient.

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