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German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), D-14558 Nuthetal, Germany and * Research Unit Nutritional Physiology "Oskar Kellner," Research Institute for the Biology of Farm Animals (FBN), 18196 Dummerstorf, Germany
3To whom correspondence should be addressed. E-mail: petzke{at}mail.dife.de.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • animal or plant food protein • amino acids • stable isotopes • hair • biomarker
Reliable biomarkers can be helpful, for example, to predict a suspected health risk for chronic diseases associated with excessive intake of meat and meat products or dietary protein (1–4). Because biomarkers do not rely on self-reporting, they are not biased by under- or overreporting of certain foodstuffs and nutrients, a frequently observed phenomenon in dietary recall methods. The underlying assumption to validate a biomarker as a measure of dietary intake is that it responds to dietary intake in a dose-dependent manner (5,6). Finally, biomarkers of food intake should be easily available and sampled noninvasively.
Hair is one of the most accessible biological materials, and its stable isotope signatures have been used to provide information about prehistoric as well as modern diet and geographical location (7–15). This is due mainly to the fact that body proteins, including hair keratin, of animals and humans reflect their dietary history by the abundance ratio of carbon and nitrogen stable isotopes, i.e., 13C/12C, and 15N/14N, respectively. In contrast to other body proteins, hair is not metabolically active, which allows a reliable recording of dietary habits during the past months. In general the body of animals is enriched in 13C and 15N relative to the diet by 
1 and 3
, respectively (16,17). The 15N abundance of tissue proteins increases within the food chain (9,18–20). Thus, animal-derived food proteins are more highly enriched in 15N than plant-derived food proteins, and the natural 15N abundance of tissue proteins can be used to differentiate between food protein preference groups (9,15).
Further, we showed that the 15N abundance in individual human plasma free and protein bound amino acids differs. For example threonine, lysine, and phenylalanine are significantly more depleted in 15N and their 15N value appears to be more stable in various populations than in BCAA or glutamic acid (21,22). This is due to differences in the degree of transamination of different amino acids (21). Thus, we hypothesize that individual amino acids with a higher degree of transamination, such as alanine or leucine, better predict animal protein intake because they better reflect the metabolic exchange of nitrogen between tissues and food nitrogen than the bulk 15N value. Considering the natural 13C signature of individual amino acids, indispensable amino acids, such as lysine, leucine, or phenylalanine, might better reflect their plant or animal origin. This is because the indispensability of amino acids is due to their carbon skeleton, which cannot be made by mammalian enzymes.
Thus, the objective of the present study was to investigate whether the bulk nitrogen and carbon isotopic composition of human hair can be used as a biomarker with which to predict the intake of animal protein, particularly meat. Further, we tested the hypothesis that individual amino acid–specific 15N and/or 13C values are better predictors of animal protein intake than bulk nitrogen and carbon isotopic abundances. To answer these questions, we studied hair samples from individuals in a large representative survey of the German population for which detailed dietary intake information was available.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For the present study, we randomly selected 50 men and 50 women, 20–50 y old, from the total study population. In addition, hair samples of the same age group were selected from participants of the VERA study who had reported that they ate no or only small quantities of meat (21 women and 6 men) in the accompanying questionnaire. This sample was added to the randomly selected group to increase the variation in animal protein intake in the study group. Stored hair samples of the selected subjects were used for carbon and nitrogen stable isotope analysis. For one of the selected women, the quantity of hair sample was insufficient for complete isotopic analysis.
Dietary data. Data about dietary habits was collected using extensive questionnaires, and food and nutrient consumption was evaluated by a 7-d dietary record using typical household measures, and, where possible, accurate weighing to assess the amounts consumed. The 7-d dietary records were coded according to the German Food Code and Nutrient Base BLS II.3 (25) and analyzed for nutrient intake.
Sample collection and preparation.
Hair samples were clipped from up to 1 cm above the scalp of the specific hair specimen collected at the survey. This section approximately corresponded to hair grown during the last 4 wk before collection and the time of dietary records. Samples (5 mg) were cut into small sections, mixed, and cleaned using a 1:1:1:1 chloroform:methanol:acetone:ether solution and agitated for 30 min to remove any lipid or shampoo residue. Solvents were removed by filtration (folded filters, Machery-Nagel), and hair samples were subsequently air dried at room temperature. Cleaned hair samples (0.3 mg) were placed in tin capsules (4 x 6 mm, IVA Analysentechnik) for determination of bulk 13C and 15N abundances.
Isotopic and amino acid analysis. The bulk 13C and 15N abundances of hair samples were determined using an elemental analyzer (EA 1108, Fisons Instruments) coupled online via a conflo interface with an isotope ratio MS (EA-IRMS; Delta C, Thermo Electron). The combustion furnace was maintained at 1020°C, and flash combustion occurred by injecting a pulse of O2 at the time of sample drop. Helium was used as carrier gas. NOx species were reduced to N2 in a reduction furnace at 650°C. Water was removed by phosphorus pentoxide in a water trap and CO2 was separated from N2 using a GC column [2 m length, 4 mm i.d., Poropak-QS (80–100 mesh), Fisons Instruments] operated isothermally at 40°C.
For isotopic analysis of individual amino acids, another aliquot of cleaned hair (1.5 mg) was hydrolyzed in 2 mL distilled 6 mol/L HCl for 24 h in PTFE capped 16 x 100 mm vials at 110°C. Hydrolysates were dried under nitrogen at 60°C and dissolved in 0.1 mol/L HCl (21,26). 13C and 15N analysis of individual amino acids was performed by GC-combustion-IRMS (GC-C-IRMS; Thermo Electron) after derivatization to their N-pivaloyl-i-propyl esters as described previously (21). The 13C abundances of amino acids measured by GC-C-IRMS were corrected by individual empirical correction factors due to the extra carbon and the reproducible isotopic fractionation introduced by the derivatization process (27).
Isotopic compositions of carbon and nitrogen in the range of natural abundance are reported in the conventional delta (
) per mil notation () (28). The 13C and 15N values of the international standards Peedee Belemnite Limestone carbonate (PDB) and atmospheric nitrogen (AIR), respectively, were assigned a value of 0.0 and can be calculated by using the following equation:
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where ISO is either 13C or 15N. Isotope ratios are Rstandard = [13C]/[12C] = 0.0112372, whereas for nitrogen Rstandard = [15N]/[14N] = 0.0036765. 13C/12C ratios are derived either from respective ratios ranging from m/z 44 to m/z 46 ions current or for 15N/14N from respective ratios of m/z 29 to m/z 28 ions current signals of the mass spectrometer. A substance with an isotope ratio larger than that of the standard (0.0) has a positive value, and is thus enriched in the heavy isotope relative to the standard.
The bulk and amino acid–specific 15N and 13C abundances as determined by EA-IRMS or GC-C-IRMS, respectively, were measured against laboratory standard gases N2 and CO2 (Linde AG) that had been calibrated against the international standards AIR and PDB, respectively. Calibration was performed against reference materials (Sucrose RM 8542 ANU, –10.47 ± 0.13 13C, National Institute of Standards and Technology; ammonium sulfate, IAEA 305A, 39.8 15N, International Atomic Energy Agency). Unmodified wheat starch (Sigma Chemical, Lot 84H0311, –23.7 13C) was used as the working standard for 13C measurements and acetanilide (Fisons Instruments, Cod. 33836700, –9.9 15N) for 15N measurements. Typical replicate measurement errors for hair samples were ± 0.2 for bulk carbon and nitrogen isotope abundances by EA-IRMS. In individual amino acids they were 0.3 and 0.5 using GC-C-IRMS and the N-pivaloyl-i-propyl derivatives for 13C and 15N, respectively.
Data processing including the correction for the 17O moiety determining 13C values was performed by the vendor-provided software ISODAT (Thermo Electron). For peak identification in the GC-C-IRMS assays, the threshold slope (slope sensitivity) for peak start and stop definitions was set to be 0.2 and 0.4 mV/s for 15N, and 0.6 and 0.6 mV/s for 13C, respectively. Integration time was 0.25 s (21). For GC-C-IRMS measurements, each sample was derivatized and analyzed in duplicate and for EA-IRMS, measurement of each sample was repeated 5 times.
Statistical analysis. Variables were tested for Gaussian distribution. Descriptive analysis includes means ± SD and the range. Comparison of means between men and women was performed with 2-sided unpaired Student’s t test. Associations between the variables were estimated by Pearson correlations (15N vs. 13C abundances, isotopic abundances vs. dietary data). Regression analysis was used to evaluate the relation between dietary data and isotope abundance. Standardized regression coefficients were tested by t test for deviation from the null hypothesis. The feasibility of the isotope abundances to predict protein intake was also tested by cross-tabulation of the observed and predicted dietary data using quintile categories. The cross-tables were analyzed regarding correctly classified subjects by the prediction equation (subjects in the diagonal categories) and subjects with grossly incorrect classification (deviation of 2 quintiles and more). The significance threshold was set at P = 0.05. We used SAS for Windows (version 8, SAS Institute) or WinSTAT® (version 1999.2, R. Fitch software) for statistical evaluation.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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15N and 13C values in human hair of the selected VERA study group were 9.8 ± 0.7 and –19.6 ± 0.4 for men (n = 56), and 9.5 ± 0.9 and –19.8 ± 0.4 for women (n = 70), respectively. The values were lower in women than in men (P < 0.04 and P < 0.006 for 15N and 13C, respectively). Additionally, the ranges were wider for women than for men by 0.9 and 0.5 for 15N and 13C, respectively.
There was a strong positive correlation between bulk 15N and 13C values in hair of the VERA study population (r = 0.386, P < 0.0001, n = 126). Further, significant positive correlations were found between bulk hair 13C values of the VERA study population and intake of indispensable amino acids such as leucine (r = 0.255, P = 0.004), lysine (r = 0.334, P = 0.0001), threonine (r = 0.297, P = 0.0007), tyrosine (r = 0.261, P = 0.003), and valine (r = 0.251, P = 0.005). The corresponding correlations between hair 15N values and intake of indispensable amino acids leucine, lysine, threonine, tyrosine, and valine were also significant (all r 
0.276, all P 
0.002). Interestingly, there was a significant positive correlation between BMI and hair 13C values (r = 0.182, P = 0.04) and 15N values (r = 0.193, P = 0.03).
To explore whether stable isotopic values of bulk hair samples could be used as a biomarker of animal protein or meat consumption, standardized regression coefficients of 13C and 15N values and their multiple R2 for variables of dietary protein and meat intake were calculated (Table 2). The isotope values strongly predicted the relative animal protein intake. Absolute animal protein intake, as well as meat intake, was significantly related to 13C and 15N values. Further, we tested the feasibility of using the 13C and 15N values to predict the intake of animal protein, relative animal protein intake, and intake of meat and processed meat by constructing cross-tables of observed and predicted values based on quintiles (Table 3). All 3 dietary intake variables could be predicted well by the hair isotope values with 10% of subjects at most deviating between "intake category" and "predicted intake category" in more than 2 quintile categories.
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0.64) percentages of animal protein intake. The references for this test were the bulk 13C and 15N values calculated according to the binary categories (Table 4). With the exception of 15N in men, the isotopic values were able to differentiate between low and high PAPI values. In contrast, the 15N and 13C abundances in individual amino acids did not have such discriminating ability across sex and isotope specific categories. Among amino acid–specific 15N abundances, a significant correlation occurred only between PAPI and valine (r = 0.203, P < 0.048), alanine (r = 0.292, P < 0.004), glycine (r = 0.305, P < 0.003), and glutamic acid (r = 0.204, P < 0.047). In addition, valine seemed to be a relatively good predictor because the 13C value of this indispensable amino acid was significantly correlated with meat (r = 0.239, P < 0.017) and protein intake (r = 0.249, P < 0.013). Interestingly, there was a significant inverse correlation between hair 15N values of alanine (r = –0.261, P < 0.010) and glycine (r = –0.227, P < 0.026) and the intake of vegetables. Comparing the values of individual amino acids in women and men who had consumed a proportion of animal protein lower or higher than 64%, we found that significant differences (P < 0.05) occurred only in women for 15N in valine, alanine, and glycine (Table 4).
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values for both 15N and 13C of individual amino acids was measured in hair samples (Table 4). Threonine was the amino acid with the lowest 15N value, whereas proline, glutamic acid, and valine had the highest 15N abundance. 15N values for phenylalanine, lysine, and histidine were higher than those for threonine, but were considerably lower than those for proline, glutamic acid, and valine. The difference between the lowest and highest 15N values of the individual amino acids threonine and proline was 25. Glycine was the amino acid with the highest 13C value, whereas threonine, the BCAA, phenylalanine, and serine had the lowest 13C abundance. The difference between the lowest and highest 13C values of the individual amino acids leucine or threonine and glycine was 30. The isotopic patterns of individual amino acids were similar in women and men (Supplemental Table 1). |
DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1 cm/mo, integrated dietary information for several months can be obtained.
Bulk 15N and 13C values were positively correlated with the daily intake of protein, meat, and animal protein and inversely with plant protein, calculated on the basis of 7-d dietary records of the selected VERA study subgroup. This was probably due to the lower 15N and 13C content of vegetable proteins compared with animal proteins as shown in previous studies (10,13,14,30,31) and confirmed for selected animal and plant proteins. Animal-derived dietary protein sources generally have greater 15N values (2–3) and 13C values (10) than plant-derived protein sources (Petzke, K. J., unpublished results). Moreover, we found that in mixed meals, the 15N values were higher (P < 0.001) when the protein content was increased by adding dietary animal protein sources. The 15N value of meals with an adequate protein content (n = 20; protein content = 11%; PAPI = 0.6) was 0.1 ± 1.7, whereas that of high protein meals (n = 20; protein content = 26%; PAPI = 0.7) was 2.2 ± 1.4 (Petzke, K. J., unpublished results). Results of 2 recent studies performed in the Okehampton area (Southwestern England) with 38 residents (15) and with 28 Oxford residents (9) indicated a relation between animal protein consumption and hair 13C and 15N abundances. However, in the Oxford study (9) ovo-lacto vegetarians (n = 6) could not be distinguished from omnivores (n = 14) on the basis of 15N values or vegans (n = 8) from both ovo-lacto vegetarians and omnivores using the 13C values. This is in contrast to the study of Bol and Pflieger (15) who reported differentiation among all 3 food preference groups (27 omnivores, 6 ovo-lacto vegetarians, 3 vegans) on the basis of both the bulk 15N and the 13C values.
The 13C range of –21.4 to –18.7 observed in the hair of our study population is characteristic for a preferential consumption of C3 plant-based protein sources, which is typical for Northwestern Europe (9,15). The 13C abundance in plant biomass depends on the type of carbon fixation reaction during photosynthesis (32,33). Consequently, different groups of plants important for human nutrition can be distinguished. These are C3 plants (wheat, barley, soy, potatoes, fruits, vegetables) with distinctly lower natural 13C abundances (range –32 to –23 13C) than C4 plants (corn, sorghum, millet, sugar cane) (range –15 and –11 13C; 18). This difference is also reflected in animal-derived food products such as milk ranging between –27 and –14 13C when the animals eat C4 plant (corn)– and C3 plant (grass and sugar beet)–based rations, respectively (34). Because Europeans still consume relatively low amounts of C4 plants and C4 plant-based food products, the 13C values of tissue components and hair are lower than those of individuals living in North America (10,14). Therefore, to assess dietary animal protein intake from the 13C and 15N isotopic signature in hair, it is important to consider the stable isotopic composition of locally available dietary proteins and foods. However, due to today’s globalization of the market, one can expect relatively similar conditions within large areas such as Northwestern Europe.
In our study, bulk 13C values of hair were as predictive of the proportion of animal protein consumption as bulk 15N values. This was not expected because previously a lower 13C shift within the food chain was observed compared with 15N abundances. In general, animal bodies are enriched in 13C and 15N relative to the diet by 1 and 3, respectively (16,17). However, the use of corn products in animal production together with the relatively high proportion of meat and meat products in the German diet (35) might explain the high predictive value of hair 13C values for animal protein intake. We assume that an indirect consumption of amino acids from corn via meat, meat, and dairy products may contribute to higher 13C values when the proportion of animal protein in the diet is high. Some of the animal-derived dietary proteins are much more positive in their 13C values than those from plant sources. This difference can be up to 16 13C comparing wheat gluten with bovine albumin (Petzke, K. J., unpublished results).
Our results confirm the high predictability of the bulk 15N values of hair for the proportion of animal protein in the diet. The animal-derived protein sources are similarly more positive in their 15N values than those from plant protein sources with the consequence that an increase in dietary animal proteins also increases the 15N value in meals (Petzke, K. J., unpublished results). These results can be explained by a relatively strong 15N shift within the food chain. Although all of the mechanisms of isotope discrimination responsible for this shift have not yet been described in detail, amino acid transamination and urea synthesis may be involved (19,36,37). It was shown in rats that hepatic nitrogen metabolism causes a discrimination of 15N isotope against 14N, resulting in a relative enrichment of 15N in body proteins and a depletion in urinary urea and ammonia by up to 10 (37).
Only a few reports exist on the 15N and 13C abundance in amino acids of different tissues and proteins at the natural abundance level. This is surprising because isotopic analysis of single amino acids could potentially allow a more detailed investigation of diet and body protein interrelations than those from bulk protein isotopic values. We previously investigated the isotopic signature of individual amino acids (21,27). However, this is the first report on human hair individual amino acids. Hair amino acids show a characteristic pattern of 15N and 13C values comparable to that in other tissue proteins of humans or animals (21,26,38). A relative depletion, e.g., in the 15N value of threonine and an enrichment in the 13C value of glycine, was also reported by others, and its meaning warrants further investigation (39,40). One of our initial hypotheses was that 15N and 13C abundances in individual hair amino acids could better predict the level of animal protein intake than those from bulk isotopic values of hair. However, we found no evidence to confirm this hypothesis. The lower predictive power using stable isotopic values of individual amino acids may be due in part to the lower precision of the GC-C-IRMS compared with EA-IRMS.
In summary, we conclude that hair 15N and 13C abundances can be used to predict the level of animal protein consumption, thereby acting as biomarkers to validate dietary assessment methods if geographical and cultural variations of food choice and isotopic composition are considered. The associations between hair 13C and 15N abundances and dietary protein intake still have to be tested in a long-term controlled dietary intervention study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supplemental Table 1 is available with the online posting of this paper at www.nutrition.org.
4 Abbreviations used: AIR, atmospheric nitrogen; EA-IRMS, elemental analysis-isotope ratio MS; GC-C-IRMS, GC-combustion-isotope ratio MS; PAPI, proportion of animal protein consumption based on 7-d dietary records; PDB, PeeDee Belemnite; VERA, nutrition survey-risk factor analysis.
Manuscript received 19 November 2004. Initial review completed 13 January 2005. Revision accepted 2 March 2005.
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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