Abstract
purpose. Lutein (L) and zeaxanthin (Z) are two dietary carotenoids that accumulate at the macula, where they are collectively known as macular pigment (MP). There is a biologically plausible rationale, with some supporting evidence, that MP may protect against age-related maculopathy (ARM). This study was undertaken to investigate the relationship between dietary intake of L and Z, serum concentrations of these carotenoids, and MP optical density in 828 healthy Irish subjects.
methods. Dietary intake of L and Z was assessed with a validated food-frequency questionnaire, and serum concentrations of these carotenoids were quantified by high-performance liquid chromatography. MP optical density was measured psychophysically, using heterochromatic flicker photometry. Demographic data, lifestyle data, and general health status, were also recorded by questionnaire, with particular attention directed toward risk-factors (established and putative) for ARM.
results. The relationships between MP optical density, serum concentrations of L (and Z), and dietary intake of L (and Z) were positive and statistically significant when analyzed for the entire study group (r = 0.136–0.303; P < 0.01 for all). Subjects with a clinically confirmed family history of ARM, current heavy cigarette smokers, subjects aged more than 53 years, and subjects with a body mass index (BMI) >27, did not demonstrate a positive and significant relationship between MP optical density and serum concentrations of Z (r = 0.041, r = 0.001, r = 0.074 and r = 0.082, respectively; P > 0.05 for all). However, there was a positive and significant relationship between MP optical density and serum concentrations of L in the presence of all these risk factors (r = 0.165 to 0.257), except for current heavy smokers (r = 0.042; P > 0.05).
conclusions. For subjects at increased risk of ARM (e.g., subjects with a clinically confirmed family history of ARM, current heavy cigarette smokers, subjects aged > 53 years and subjects with a BMI > 27) retinal capture and/or retinal stabilization of Z appears to be compromised, whereas retinal uptake and/or stabilization of L appears to be compromised in current heavy smokers only. Given the lack of MP in association with risk for ARM, the findings indicate that a retina predisposed to this condition may have an impaired ability to accumulate circulating Z.
Age-related macular degeneration (AMD), the late stage of age-related maculopathy (ARM), is the leading cause of blindness in individuals more than 65 years of age in the Western World.
1 2 The etiological mechanisms underlying ARM continue to elude, but there is a growing body of evidence implicating oxidative stress and/or cumulative blue light damage in the process.
3 The carotenoids lutein (L) and zeaxanthin (Z), to the exclusion of all other carotenoids, are concentrated in the macula, where they are collectively referred to as macular pigment (MP).
MP is a blue-light filter at a prereceptorial level,
4 and demonstrates powerful antioxidant properties.
5 Consequently, it is believed that MP may protect against macular diseases attributable to oxidative stress, most notably ARM. The hypothesized protective effect of MP for ARM is rendered all the more provocative by its dietary origins.
Existing evidence in support of the view that MP protects against ARM is dominated by cross-sectional and epidemiologic reports. In brief, this evidence refers to parallels between risk for ARM and a relative lack of L and/or Z in the diet and/or serum and/or macula (SanGiovanni JP et al.
IOVS 2004;45:ARVO E-Abstract 2242).
6 7
Any protective effect of MP for ARM is premised on its defense against chronic and cumulative oxidative and/or photochemical damage, and, as such, would need to be exerted over a long period and decades before the onset of disease.
3 Given that a positive and significant relationship between MP and its constituent carotenoids in the diet and in the serum has been consistently demonstrated in healthy subjects
8 9 10 11 12 13 14 and that a relative lack of MP has been reported in association with certain risk-factors for ARM (SanGiovanni JP et al.
IOVS 2004;45:ARVO E-Abstract 2242),
6 7 it would seem logical to investigate whether the relationship between dietary and serum levels of these carotenoids, or whether the relationship between serum concentrations of L (and/or Z) and MP, is influenced by these risk-factors in young and middle-aged subjects. In other words, and for example, is tobacco use (an established risk-factor for ARM) associated with an attenuated relationship between serum levels of L and MP optical density? And if so, can the data indicate whether such a finding represents compromised retinal capture of circulating carotenoids among smokers or an altered stabilization/utilization of this xanthophyll within the retina of those who consume tobacco? To date, the influence of risk factors for ARM on these relationships has not been investigated, with the exception of the influence of sex.
15 However, that study reported the concentrations of L and Z collectively rather than individually.
The present study was designed to investigate the relationship between dietary intake of L and Z, serum concentrations of these carotenoids, and MP optical density, and to relate the findings to risk factors for ARM (both putative and established), in 828 healthy Irish subjects aged 20 to 60 years.
Eight hundred twenty-eight healthy subjects from an Irish population were enrolled to participate in the study, which was authorized by the Research Ethics Committee of the Waterford Institute of Technology. Informed consent was obtained from each subject, and the experimental procedures adhered to the tenets of the Declaration of Helsinki. The inclusion criteria were white race and age between 20 and 60 years, and exclusion criteria comprised any ocular disease, visual acuity less than 6/18 in both eyes, or pregnancy.
Subjects were recruited by one of two means. The majority of subjects were recruited as a result of posters, newsletters, and word of mouth in the local community (group 1: n = 646). Also, patients attending the Department of Ophthalmology at Waterford Regional Hospital who had ARM (early and/or late) were encouraged to invite their offspring to participate (group 2: n = 182). For a subject recruited by posters and/or word of mouth who thought he or she had a direct family history of ARM (offspring of ARM sufferers), the medical records for that subject’s appropriate parent were examined by a trained ophthalmologist, and if there was a positive family history of ARM, confirmatory documentation was secured, and that subject was placed into the group referred to as having a clinically confirmed family history of ARM. Of the 828 subjects who were recruited in total, we were unable to obtain MP measurements in 28 of these (coefficient of variation [CoV] >20%; n = 20; and visual acuity <6/18 in both eyes; n = 8). In addition, we failed to obtain dietary data from 2 individuals and serum data from 10 individuals. Also, demographic data from two individuals was misplaced.
Blood samples (6–8 mL) were collected from all subjects on the same day as the dietary and MP optical density analysis. Serum was separated from blood by centrifugation at 5000 rpm for 10 minutes and then aliquoted into three light-sensitive microcentrifuge tubes and stored at −70°C until time of analysis. Duplicate extractions were performed for each serum sample. A 0.4-mL aliquot of serum was pipetted into a light-sensitive microcentrifuge tubes (2 mL total capacity). Ethanol (0.30 mL) containing 0.25 g/L butyrated hydroxytoluene (BHT) and internal standard (tocopherol acetate) was added to each tube. Heptane (0.5 mL) was then added, and samples were vortexed vigorously for 1 minute followed by centrifugation at 2000 rpm for 5 minutes (MSC Micro Centaur; Davison & Hardy Ltd., Belfast, UK). The resultant heptane layer was retained and transferred to a second labeled light-sensitive microcentrifuge tube, and a second heptane extraction was performed. The combined heptane layers were immediately evaporated to dryness under nitrogen. These dried samples were reconstituted in methanol (200 μL), and 150 μL was injected for high-performance liquid chromatography (HPLC) analysis.
We used an HPLC system (Hewlett-Packard HP 1090 LC; Agilent, Dublin, Ireland) with photodiode array detection. A 5-μm analytical/preparative 4.6 × 250-mm specialized reversed-phase column (201TP; Vydac, Hesperia, CA) was used with an in-line guard column. The mobile phase consisted of 97% methanol and 3% tetrahydrofuran. The flow rate was 1 mL/min, and the total run time was 15 minutes. All carotenoid peaks were integrated and quantified (Chem Station software; Agilent).
DSM Nutritional Products (Basel, Switzerland) provided the L and Z standards that were used to generate standard curves for quantification of these carotenoids. This assay was validated against the National Institute of Standards and Technology (NIST) Standard before analysis.
Sex.
Family History Status.
Cigarette Smoking.
Age.
Body Mass Index.
Sex.
Family History.
Cigarette Smoking.
Age.
Body Mass Index.
This study was specifically designed to report on the relationships between dietary intake of L and Z, serum concentrations of L and Z, and MP optical density, and to relate the findings to risk for ARM in 828 healthy Irish subjects aged 20 to 60 years.
Of the eight published observational studies that have examined the relationship between dietary antioxidants and risk for ARM, four have found a protective effect associated with a high intake of carotenoids.
7 28 29 30 31 32 33 34 35 Also, a recent report by the Age-Related Eye Disease Study group has shown that a higher dietary intake of L and Z is associated with a decreased likelihood of the development of advanced ARM (SanGiovanni JP et al.
IOVS 2004;45:ARVO E-Abstract 2242).
Mean daily intake of L and Z combined, varies from 0.8 mg to 4 mg per day, depending on the population studied and the method of dietary assessment used.
36 37 Further, daily intake of L has been shown to vary widely among individuals, as illustrated by a standard deviation of 2.45 mg/d in a recently published study.
38 In the 826 volunteers who completed FFQs in our study, mean dietary intake (±SD) of L and Z was 1.399 ± 0.79 mg/d and 0.199 ± 0.117 mg/d, respectively. These values are comparable with those obtained by previous investigators (using similar dietary assessment techniques) in similar age groups.
10 11 27 39 40 41 42
Most previous studies in which the relationship between serum concentrations of L (and Z) and risk for ARM have been examined, have reported the concentrations of these two carotenoids collectively rather than individually.
6 7 43 44 45 46 47 48 49 Of these, only one found a significant association between low serum concentrations of L and Z (combined) and risk for ARM.
46 Recently, however, Gale et al.
6 investigated the relationship between serum L and Z (separately) and risk for ARM and found that ARM was associated with a relative lack of serum Z (but not L). It is possible that the analysis of serum L and Z in a collective fashion in those previous studies, rather than investigating these carotenoids separately, had masked the relative importance of serum Z concentrations. In our study, mean serum levels (±SD) of L and Z were 0.087 ± 0.042 μg/mL and 0.026 ± 0.016 μg/mL, respectively, which are consistent with those obtained by previous investigators for similar age groups.
10 47 50 51 52
Of the nine observational studies analyzing the relationship between dietary intake of L and Z and serum concentrations of these carotenoids, all have demonstrated significant and positive relationships (
r = 0.21–0.74;
P < 0.05 for all).
10 11 12 13 15 40 41 42 53 The largest of these studies included 2786 subjects, and found that every 10% increase in estimated dietary intake of L and Z was associated with a 2.4% increase in serum L concentration.
16 Similarly, we found a positive and significant relationship between the absolute dietary intake of L and Z, and serum concentrations of the respective carotenoids (
r = 0.280 and 0.237, respectively). Of note, after adjustment for confounding variables, the relationships were only slightly enhanced (absolute diet L/serum L,
r = 0.284 and absolute diet Z/serum Z,
r = 0.250).
Also, energy-adjusted (by the residuals method) and nutrient density L (and Z) were positively and significantly related to serum concentrations of their respective carotenoid, with Pearson correlation coefficients ranging from 0.259 to 0.303 for the entire study group. We felt it appropriate to calculate energy-adjusted values, as an absolute amount of a specific nutrient tends to have less of an effect for a larger, higher energy-consuming person, than for a smaller person. The slightly stronger relationships we found for energy-adjusted and nutrient density values most likely reflect this.
Further, the present study comprised a sample size large enough to assess and compare, for the first time, the relationships between dietary intake of L (and Z), serum concentrations of L (and Z) and MP optical density, among different groups in the normal population (e.g., males, females, subjects with a confirmed family history of ARM, subjects with no known family history of ARM, cigarette smokers, nonsmokers).
Of interest, subdividing according to sex, family history, cigarette smoking, age, and BMI (putative and known risk factors for ARM), the respective relationships between dietary intake and serum concentrations of L (and Z) remained positive and statistically significant, with the sole exception of the relationship between dietary (whether absolute, energy-adjusted or nutrient densities) and serum levels of L for current heavy smokers (≥20 cigarettes per day), who failed to exhibit a significant relationship between these measures
(Table 5) .
It appears, therefore, that the risk that sex, family history of ARM, smoking, age, and BMI represent for ARM is not attributable to impaired digestion and/or intestinal absorption of Z. Similarly, the risk that sex, family history of ARM, age, and BMI represent for ARM is not associated with impaired digestion or intestinal absorption of L. However, the failure of heavy cigarette smokers to exhibit the typical positive and significant relationship between dietary intake of L and serum concentrations of this carotenoid is interesting and warrants discussion.
Given that heavy cigarette smokers typically had a dietary intake of L similar to that of light cigarette smokers and nonsmokers (P > 0.05, for all), and that heavy cigarette smokers had lower serum levels of L than did nonsmokers (even after adjustment for dietary intake of L), indicates that heavy users of tobacco have a compromised digestion and/or absorption of this carotenoid or that consumption of cigarettes reduces circulating levels of L.
Possible mechanisms whereby tobacco use is related to reduced circulation of serum L and/or an impaired diet L/serum L relationship requires discussion. One possibility includes an altered lipoprotein profile in cigarette smokers, with a consequential impact on the transport of L within serum. For example, high-density lipoproteins (HDL) are known to be the primary carriers of L and Z, whereas low-density lipoproteins (LDL) transport hydrocarbon carotenoids (e.g., lycopene, β-carotene).
54 Given that cigarette smokers are known to have significantly reduced levels of HDL when compared to nonsmokers,
55 it is possible that reduced HDL in tobacco users may account for the reduced serum concentrations of L (and Z) that we report for such individuals. Alternatively, the increased circulating pro-oxidant load in cigarette smokers could result in depletion of circulating levels of L (and Z). Indeed, Handelman et al.
56 have shown that exposing human plasma to the gas phase of cigarette smoke results in a significant depletion (up to 60%) of L (+Z) after just 9 hours’ exposure time (9 hours’ exposure time = 3 puffs/h for 9 hours).
Studies investigating the relationship between retinal and serum carotenoids should be interpreted with caution, primarily because serum concentrations of L and Z reflect recent nutritional intake only.
57 In contrast, MP has a slow biological turnover, and probably reflects the local balance between pro-oxidant stresses and antioxidant defenses in the retina. In other words, a dramatic change in diet is unlikely to affect MP for several weeks, but will be reflected in much more rapid changes in serum concentrations of L and Z.
Of the nine observational studies investigating the relationship between serum L and Z and MP optical density, seven have found positive and significant correlations (
r = 0.21–0.82,
P < 0.05).
10 12 13 15 39 42 50 58 59 Of the studies that did not report significant relationships, one was limited by its inclusion of only 20 subjects (10 monozygotic twin pairs)
59 and the other by its narrow range of serum concentrations of L and Z, represented by standard deviations of 0.043 μg/mL and 0.014 μg/mL, respectively.
58
We also found serum concentrations of L and Z to be positive and significant predictors of MP optical density. Of interest, controlling for age, sex, family history, cigarette smoking, and absolute dietary intake of L, enhanced the strength of the relationship between serum L and MP optical density (r = 0.232, P < 0.01), whereas, the strength of the relationship between serum Z and MP optical density was reduced after controlling for the same variables (and replacing absolute dietary intake of L with that of Z; r = 0.082, P < 0.05).
Finally, and of noteworthy interest, subjects with a clinically confirmed family history of ARM, current heavy cigarette smokers, subjects >53 years of age, and subjects with a BMI > 27, did not exhibit a significant relationship between MP optical density and serum concentrations of Z (r = 0.041, r = 0.001, r = 0.074 and r = 0.082, respectively, P > 0.05, for all). Similarly, current heavy cigarette smokers did not exhibit a positive and significant relationship between serum L and MP optical density (r = 0.042, P > 0.05). For all other subgroups (men, women, subjects with no known family history of ARM, nonsmokers, current light smokers, subjects aged <31 years, subjects between 31 and 52 years of age, subjects with a BMI <23, and subjects with a BMI between 23 and 27), the relationship between MP optical density and serum concentrations of L (and Z) were positive and statistically significant.
It appears, therefore, that risk factors for ARM are associated with a lack of the typical positive and significant relationship between serum concentrations of Z and MP optical density before the onset of disease. Although many of these risk factors were associated with lower levels of Z in serum and with lower MP optical density, it is the attenuated relationship between these variables that is of interest, and warrants discussion. The lack of a positive and significant relationship between serum Z and MP optical density could be attributable to defective capture of this carotenoid by the retina and/or impaired stabilization within the retina, in association with risk for ARM. Certainly, high BMI could be associated with competition between the retina and adipose tissue for circulating carotenoids, but it is difficult to explain why uptake of Z (and not L) by the retina may be compromised in association with other risk factors for ARM.
However, the mechanisms governing retinal capture of carotenoids remain poorly understood, and xanthophyll-binding proteins (XBPs) are thought to be important.
60 Of interest, a zeaxanthin-binding protein (ZBP; Pi isoform of glutathione S-transferase [GSTP1]), which is found in high concentrations of the inner and outer plexiform layers of the macula (location of MP),
4 61 has been shown to demonstrate a high affinity for dietary Z, with poor affinity for L.
62 This finding indicates that L and Z have unique specific binding proteins within the macula, which are responsible for uptake and/or stabilization of each carotenoid. It is possible, in theory at least, that some risk factors for ARM, such as family history of ARM and age, could be associated with a lack of GSTP1, and thus explain our findings.
Alternatively, however, it is possible that our finding that risk for ARM is not associated with the typical significant and positive relationships between MP and serum concentrations of Z, and the relative lack of MP and serum Z seen in association with many of these risk factors, reflects the compromised ability of the retina to stabilize this carotenoid. For example, there is a growing body of evidence that oxidative stress is etiologically important in the pathogenesis of ARM, and it is known that Z acts as an antioxidant within the retinal tissues. It is reasonable to hypothesize, therefore, that a retina at particularly high risk of ARM represents a high oxidative stress environment, and would be associated with excessive depletion of Z, and a consequentially reduced MP optical density and an attenuated relationship, if any, with serum levels of this carotenoid.
Of note, our finding that serum concentrations of Z (adjusted for dietary intake) were significantly reduced in association with three established risk factors for this condition (age, tobacco use, and BMI) in healthy young and middle-aged subjects, is consistent with the finding of Gale et al.
6 that ARM is associated with a relative lack of serum Z. With respect to our finding that heavy cigarette smokers also did not demonstrate a positive and significant relationship between MP optical density and serum levels of L, we offer the same explanation as for Z.
However, the fact that MP represents a composite measure of retinal L, Z, and meso-Z may confound any interpretation of our findings. For example, Z dominates the central foveal region, whereas L is typically dominant in the perifoveal region.
61 63 meso-Z [(3
R,3′
S)-β,β-carotene-3,3′diol], a carotenoid not found in the normal human diet, is observed to reach its maximum concentration at the foveola, at the same point where the L to Z ratio reaches a minimum.
61 64 65 Of interest, it has been shown that the L proportion of MP decreases with increasing proportion of meso-Z. It has been suggested that L undergoes a chemical oxidation in the central retina (double bond isomerization), and is oxidized to meso-Z.
5 64 Such a conversion, within the retina, may cause changes in the anatomic distribution of MP. For example, a person with large amounts of serum L might be expected to have high peak MP (as this is where meso-Z is found in the retina). Therefore, the relationship between MP and each of its two constituent carotenoids within serum will be influenced by the amount of L that is converted to
meso-Z at the precise retinal location where MP optical density is being measured. However, a detailed in vitro study would be needed to explore such an effect. Of note, there is no evidence to suggest that metabolic transformations involving L and Z (L to
meso-Z) occur in serum.
64 Consistent with this view, a recent study in which rhesus monkeys, reared on carotenoid-free diets, were fed L or Z reported an absence of serum Z in the L-fed group and only trace amounts of 3′didehydrolutein (a variant of L) in the Z-fed group, suggesting that such interconversion between L and Z in the serum is negligible.
66 In any case, all in vivo techniques that measure MP do so without distinguishing between retinal L, Z, or
meso-Z (because the optical properties are identical for these three compounds).
In conclusion, there is no demonstrable relationship between serum levels of Z and MP optical density in heavy cigarette smokers, subjects with a family history of ARM, older subjects and subjects with a BMI > 27, and no demonstrable relationship between serum L and MP optical density in heavy smokers. The finding that risk for ARM is not associated with the typical positive and significant relationship between serum Z and MP optical density in subjects without such risk factors, and decades before the onset of disease, would be consistent with compromised retinal capture and/or stabilization of this carotenoid in maculae predisposed to ARM.
Supported by Fighting Blindness Ireland.
Submitted for publication July 26, 2006; revised September 6, 2006; accepted November 22, 2006.
Disclosure:
J.M. Nolan, None;
J. Stac k, None;
E. O’Connell, None;
S. Beatty, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: John M. Nolan, Macular Pigment Research Group, Waterford Institute of Technology, Cork Road, Waterford, Ireland;
[email protected].
Table 1. Pearson Correlation Matrix Showing Relationships between the Study Parameters for the Entire Study Group
Table 1. Pearson Correlation Matrix Showing Relationships between the Study Parameters for the Entire Study Group
| Absolute Dietary L (mg/day) | Energy-Adjusted Dietary L | Nutrient Density of Dietary L | Serum L (μg/mL) | Absolute Dietary Z (mg/day) | Energy-Adjusted Dietary Z | Nutrient Density of Dietary Z | Serum Z (μg/mL) | MP Optical Density |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.963* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.848* | 0.928* | 1 | | | | | | |
| Serum L (μg/mL) | 0.280* | 0.303* | 0.299* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.697* | 0.609* | 0.479* | 0.169* | 1 | | | | |
| Energy-adjusted dietary Z | 0.664* | 0.668* | 0.603* | 0.206* | 0.912* | 1 | | | |
| Nutrient density of dietary Z | 0.585* | 0.643* | 0.677* | 0.231* | 0.805* | 0.939* | 1 | | |
| Serum Z (μg/mL) | 0.160* | 0.166* | 0.146* | 0.462* | 0.237* | 0.260* | 0.259* | 1 | |
| MP optical density | 0.208* | 0.182* | 0.136* | 0.181* | 0.218* | 0.184* | 0.166* | 0.166* | 1 |
Table 2. Pearson Correlation Matrix Showing Relationships between the Study Parameters for the Entire Study Group after Removing Subjects with Unrealistic Energy Values
Table 2. Pearson Correlation Matrix Showing Relationships between the Study Parameters for the Entire Study Group after Removing Subjects with Unrealistic Energy Values
| Absolute Dietary L (mg/day) | Energy-Adjusted Dietary L | Nutrient Density of Dietary L | Serum L (μg/mL) | Absolute Dietary Z (mg/day) | Energy-Adjusted Dietary Z | Nutrient Density of Dietary Z | Serum Z (μg/mL) | MP Optical Density |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.984* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.889* | 0.938* | 1 | | | | | | |
| Serum L (μg/mL) | 0.286* | 0.301* | 0.300* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.682* | 0.637* | 0.529* | 0.189* | 1 | | | | |
| Energy-adjusted dietary Z | 0.658* | 0.637* | 0.615* | 0.216* | 0.953* | 1 | | | |
| Nutrient density of dietary Z | 0.608* | 0.669* | 0.678* | 0.238* | 0.862* | 0.951* | 1 | | |
| Serum Z (μg/mL) | 0.150* | 0.645* | 0.143* | 0.455* | 0.249* | 0.254* | 0.258* | 1 | |
| MP optical density | 0.198* | 0.153* | 0.137* | 0.170* | 0.203* | 0.175* | 0.154* | 0.160* | 1 |
Table 3. Pearson Correlation Matrix Showing Relationships between the Study Parameters, Analyzed Separately for Men and Women
Table 3. Pearson Correlation Matrix Showing Relationships between the Study Parameters, Analyzed Separately for Men and Women
| Absolute Dietary L (mg/day) | Energy-Adjusted Dietary L | Nutrient Density of Dietary L | Serum L (μg/mL) | Absolute Dietary Z (mg/day) | Energy-Adjusted Dietary Z | Nutrient Density of Dietary Z | Serum Z (μg/mL) | MP Optical Density |
| Men (n = 288) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.937* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.871* | 0.950* | 1 | | | | | | |
| Serum L (μg/mL) | 0.201* | 0.300* | 0.263* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.651* | 0.554* | 0.521* | 0.136* | 1 | | | | |
| Energy-adjusted dietary Z | 0.560* | 0.598* | 0.586* | 0.147* | 0.926* | 1 | | | |
| Nutrient density of dietary Z | 0.464* | 0.529* | 0.578* | 0.133* | 0.846* | 0.950* | 1 | | |
| Serum Z (μg/mL) | 0.201* | 0.217* | 0.178* | 0.448* | 0.293* | 0.319* | 0.295* | 1 | |
| MP optical density | 0.261* | 0.234* | 0.194* | 0.195* | 0.236* | 0.206* | 0.165* | 0.164* | 1 |
| Women (n = 538) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.955* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.854* | 0.938* | 1 | | | | | | |
| Serum L (μg/mL) | 0.271* | 0.292* | 0.299* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.718* | 0.602* | 0.476* | 0.179* | 1 | | | | |
| Energy-adjusted dietary Z | 0.658* | 0.688* | 0.624* | 0.219* | 0.874* | 1 | | | |
| Nutrient density of dietary Z | 0.633* | 0.681* | 0.699* | 0.255* | 0.797* | 0.946* | 1 | | |
| Serum Z (μg/mL) | 0.139* | 0.139* | 0.133* | 0.449* | 0.211* | 0.230* | 0.241* | 1 | |
| MP optical density | 0.207* | 0.196* | 0.167* | 0.195* | 0.221* | 0.217* | 0.205* | 0.176* | 1 |
Table 4. Pearson Correlation Matrix Showing Relationships between the Study Parameters, Analyzed Separately for Subjects with and without a Clinically Confirmed Family History of ARM
Table 4. Pearson Correlation Matrix Showing Relationships between the Study Parameters, Analyzed Separately for Subjects with and without a Clinically Confirmed Family History of ARM
| Absolute Dietary L (mg/day) | Energy-Adjusted Dietary L | Nutrient Density of Dietary L | Serum L (μg/mL) | Absolute Dietary Z (mg/day) | Energy-Adjusted Dietary Z | Nutrient Density of Dietary Z | Serum Z (μg/mL) | MP Optical Density |
| No known family history of ARM (n = 644) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.962* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.861* | 0.944* | 1 | | | | | | |
| Serum L (μg/mL) | 0.239* | 0.251* | 0.241* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.684* | 0.595* | 0.473* | 0.154* | 1 | | | | |
| Energy-adjusted dietary Z | 0.619* | 0.642* | 0.584* | 0.169* | 0.916* | 1 | | | |
| Nutrient density of dietary Z | 0.538* | 0.592* | 0.604* | 0.182* | 0.821* | 0.949* | 1 | | |
| Serum Z (μg/mL) | 0.141* | 0.141* | 0.118* | 0.492* | 0.241* | 0.256* | 0.259* | 1 | |
| MP optical density | 0.230* | 0.199* | 0.174* | 0.210* | 0.228* | 0.186* | 0.174* | 0.209* | 1 |
| Clinically confirmed family history of ARM (n = 182) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.996* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.842* | 0.916* | 1 | | | | | | |
| Serum L (μg/mL) | 0.364* | 0.413* | 0.394* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.748* | 0.660* | 0.526* | 0.210* | 1 | | | | |
| Energy-adjusted dietary Z | 0.723* | 0.751* | 0.681* | 0.308* | 0.900* | 1 | | | |
| Nutrient density of dietary Z | 0.708* | 0.769* | 0.836* | 0.344* | 0.764* | 0.916* | 1 | | |
| Serum Z (μg/mL) | 0.231* | 0.260* | 0.242* | 0.377* | 0.216* | 0.279* | 0.266* | 1 | |
| MP optical density | 0.240* | 0.221* | 0.149, † | 0.257* | 0.228* | 0.205* | 0.167, † | 0.041, ‡ | 1 |
Table 5. Pearson Correlation Matrix Showing Relationships between the Study Parameters, Analyzed According to Smoking Status
Table 5. Pearson Correlation Matrix Showing Relationships between the Study Parameters, Analyzed According to Smoking Status
| Absolute Dietary L (mg/day) | Energy-Adjusted Dietary L | Nutrient Density of Dietary L | Serum L (μg/mL) | Absolute Dietary Z (mg/day) | Energy-Adjusted Dietary Z | Nutrient Density of Dietary Z | Serum Z (μg/mL) | MP Optical Density |
| Nonsmokers (n = 660) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.966* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.858* | 0.932* | 1 | | | | | | |
| Serum L (μg/mL) | 0.265* | 0.283* | 0.282* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.681* | 0.595* | 0.482* | 0.173* | 1 | | | | |
| Energy-adjusted dietary Z | 0.634* | 0.658* | 0.606* | 0.205* | 0.911* | 1 | | | |
| Nutrient density of dietary Z | 0.585* | 0.637* | 0.677* | 0.231* | 0.813* | 0.944* | 1 | | |
| Serum Z (μg/mL) | 0.103* | 0.108* | 0.103* | 0.437* | 0.218* | 0.241* | 0.248* | 1 | |
| MP optical density | 0.200* | 0.178* | 0.145* | 0.165* | 0.211* | 0.182* | 0.167* | 0.165* | 1 |
| Current light smokers <20 cigarettes per day (n = 120) | | | | | | | | | |
| Energy-adjusted dietary L | 0.958* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.896* | 0.912* | 1 | | | | | | |
| Serum L (μg/mL) | 0.431* | 0.488* | 0.488* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.756* | 0.665* | 0.477* | 0.211* | 1 | | | | |
| Energy-adjusted dietary Z | 0.679* | 0.701* | 0.595* | 0.268* | 0.920* | 1 | | | |
| Nutrient density of dietary Z | 0.554* | 0.628* | 0.653* | 0.293* | 0.770* | 0.920* | 1 | | |
| Serum Z (μg/mL) | 0.406* | 0.426* | 0.385* | 0.501* | 0.300* | 0.320* | 0.297* | 1 | |
| MP optical density | 0.323* | 0.311* | 0.188† | 0.224* | 0.304* | 0.294* | 0.226* | 0.194† | 1 |
| Current heavy smokers ≥20 cigarettes per day (n = 46) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.941* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.862* | 0.961* | 1 | | | | | | |
| Serum L (μg/mL) | 0.154‡ | 0.153‡ | 0.154‡ | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.766* | 0.677* | 0.569* | 0.085 | 1 | | | | |
| Energy-adjusted dietary Z | 0.678* | 0.745* | 0.688* | 0.079 | 0.897* | 1 | | | |
| Nutrient density of dietary Z | 0.704* | 0.289* | 0.752* | 0.101 | 0.873* | 0.983* | 1 | | |
| Serum Z (μg/mL) | 0.275* | 0.426* | 0.263* | 0.674* | 0.291* | 0.313* | 0.308* | 1 | |
| MP optical density | 0.019‡ | −0.134‡ | −0.165‡ | 0.042‡ | 0.094‡ | −0.104‡ | −0.138‡ | 0.001‡ | 1 |
Table 6. Pearson Correlation Matrix Showing Relationships between Study Parameters, Analyzed Separately for Subjects <31 Years, between 31 and 53 Years, and >53 Years
Table 6. Pearson Correlation Matrix Showing Relationships between Study Parameters, Analyzed Separately for Subjects <31 Years, between 31 and 53 Years, and >53 Years
| Absolute Dietary L (mg/day) | Energy-Adjusted Dietary L | Nutrient Density of Dietary L | Serum L (μg/mL) | Absolute Dietary Z (mg/day) | Energy-Adjusted Dietary Z | Nutrient Density of Dietary Z | Serum Z (μg/mL) | MP Optical Density |
| Subjects aged <31 years (n = 195) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.966* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.871* | 0.943* | 1 | | | | | | |
| Serum L (μg/mL) | 0.310* | 0.333* | 0.313* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.762* | 0.687* | 0.542* | 0.206* | 1 | | | | |
| Energy-adjusted dietary Z | 0.715* | 0.735* | 0.639* | 0.239* | 0.931* | 1 | | | |
| Nutrient density of dietary Z | 0.648* | 0.702* | 0.667* | 0.250* | 0.847* | 0.951* | 1 | | |
| Serum Z (μg/mL) | 0.102* | 0.134* | 0.138* | 0.513* | 0.191* | 0.210* | 0.226* | 1 | |
| MP optical density | 0.170† | 0.157* | 0.153† | 0.233* | 0.147* | 0.133† | 0.233* | 0.197* | 1 |
| Subjects aged 31 to 53 years (n = 435) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.962* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.844* | 0.925* | 1 | | | | | | |
| Serum L (μg/mL) | 0.295* | 0.307* | 0.311* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.665* | 0.576* | 0.469* | 0.164* | 1 | | | | |
| Energy-adjusted dietary Z | 0.610* | 0.645* | 0.607* | 0.185* | 0.899* | 1 | | | |
| Nutrient density of dietary Z | 0.569* | 0.633* | 0.701* | 0.223* | 0.790* | 0.933* | 1 | | |
| Serum Z (μg/mL) | 0.160* | 0.226* | 0.144* | 0.473* | 0.241* | 0.257* | 0.253* | 1 | |
| MP optical density | 0.239* | 0.237* | 0.192* | 0.237* | 0.233* | 0.193* | 0.160* | 0.137† | 1 |
| Subjects aged >53 years (n = 196) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.970* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.866* | 0.941* | 1 | | | | | | |
| Serum L (μg/mL) | 0.260* | 0.283* | 0.262* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.693* | 0.614* | 0.490* | 0.247* | 1 | | | | |
| Energy-adjusted dietary Z | 0.628* | 0.646* | 0.596* | 0.293* | 0.917* | 1 | | | |
| Nutrient density of dietary Z | 0.548* | 0.597* | 0.621* | 0.294* | 0.818* | 0.951* | 1 | | |
| Serum Z (μg/mL) | 0.249* | 0.266* | 0.249* | 0.563* | 0.252* | 0.288* | 0.305* | 1 | |
| MP optical density | 0.189* | 0.198* | 0.195* | 0.177† | 0.198* | 0.224* | 0.233* | 0.074‡ | 1 |
Table 7. Pearson Correlation Matrix Showing Relationships between Study Parameters, Analyzed for Subjects with a BMI <23, with a BMI between 23 and 27, and with a BMI >27
Table 7. Pearson Correlation Matrix Showing Relationships between Study Parameters, Analyzed for Subjects with a BMI <23, with a BMI between 23 and 27, and with a BMI >27
| Absolute Dietary L (mg/day) | Energy-Adjusted Dietary L | Nutrient Density of Dietary L | Serum L (μg/mL) | Absolute Dietary Z (mg/day) | Energy-Adjusted Dietary Z | Nutrient Density of Dietary Z | Serum Z (μg/mL) | MP Optical Density |
| Subjects with BMI <23 (n = 270) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.970* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.834* | 0.913* | 1 | | | | | | |
| Serum L (μg/mL) | 0.302* | 0.325* | 0.294* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.781* | 0.708* | 0.526* | 0.204* | 1 | | | | |
| Energy-adjusted dietary Z | 0.734* | 0.751* | 0.640* | 0.237* | 0.930* | 1 | | | |
| Nutrient density of dietary Z | 0.664* | 0.720* | 0.724* | 0.263* | 0.814* | 0.940* | 1 | | |
| Serum Z (μg/mL) | 0.151† | 0.154* | 0.103* | 0.469* | 0.209* | 0.225* | 0.220* | 1 | |
| MP optical density | 0.317* | 0.278* | 0.194* | 0.181* | 0.358* | 0.309* | 0.250* | 0.230* | 1 |
| Subjects with BMI 23–27 (n = 345) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.959* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.830* | 0.920* | 1 | | | | | | |
| Serum L (μg/mL) | 0.320* | 0.349* | 0.351* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.699* | 0.596* | 0.463* | 0.159* | 1 | | | | |
| Energy-adjusted dietary Z | 0.640* | 0.659* | 0.598* | 0.201* | 0.906* | 1 | | | |
| Nutrient density of dietary Z | 0.595* | 0.649* | 0.695* | 0.247* | 0.800* | 0.940* | 1 | | |
| Serum Z (μg/mL) | 0.189† | 0.191† | 0.164* | 0.411* | 0.238* | 0.257* | 0.253* | 1 | |
| MP optical density | 0.193† | 0.157† | 0.113† | 0.198† | 0.164* | 0.112† | 0.155† | 0.194† | 1 |
| Subjects with BMI >27 (n = 211) | | | | | | | | | |
| Absolute dietary L (mg/day) | 1 | | | | | | | | |
| Energy-adjusted dietary L | 0.959* | 1 | | | | | | | |
| Nutrient density of dietary L | 0.881* | 0.957* | 1 | | | | | | |
| Serum L (μg/mL) | 0.171* | 0.179* | 0.199* | 1 | | | | | |
| Absolute dietary Z (mg/day) | 0.578* | 0.492* | 0.428* | 0.11 | 1 | | | | |
| Energy-adjusted dietary Z | 0.513* | 0.561* | 0.553* | 0.123 | 0.890* | 1 | | | |
| Nutrient density of dietary Z | 0.437* | 0.502* | 0.553* | 0.127 | 0.810* | 0.946* | 1 | | |
| Serum Z (μg/mL) | 0.118* | 0.133* | 0.185* | 0.484* | 0.217* | 0.249* | 0.273* | 1 | |
| MP optical density | 0.136† | 0.112‡ | 0.083‡ | 0.165* | 0.201* | 0.168* | 0.155* | 0.082‡ | 1 |
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