Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Urinary C-Peptide Excretion in Free-Living Healthy Children Is Related to Dietary Carbohydrate Intake but Not to the Dietary Glycemic Index1 Anette E. Buyken,2 Yvonne Kellerhoff, Sebastian Hahn, Anja Kroke, and Thomas Remer Research Institute of Child Nutrition, Dortmund, Germany ABSTRACT This study first examined whether urinary C-peptide (UCP), stored at 208C, remains stable over the long term, and second, whether the dietary glycemic index (GI) and the glycemic load (GL: GI 3 g carbohydrates) are related to the 24-h UCP excretion of healthy children. Participants in the Dortmund Nutrition and Anthropometric Longitudinally Designed Study with 24-h urine collections and a simultaneously completed weighed dietary record were included. From these, 3 comparable groups of 7- to 8-y-old children (n ¼ 40 each) from 1990, 1996, and 2002 were randomly selected (total n ¼ 120). C-peptide was measured with a 1-site ELISA. A GI value was assigned to all recorded foods containing carbohydrates (CHO). Statistical equivalence tests corroborated that UCP excretion in the 3 sampling periods was equivalent when corrected for body weight and protein intake (P , 0.05). UCP excretion was associated with the GL after adjustment for body weight, protein, and fiber intake [mean UCP (95% CI) in GL tertiles 1–3: 6.19 (5.37, 7.14) vs. 7.82 (6.77, 9.02) vs. 7.76 (6.71, 8.97) nmol/d, P for difference 0.04]. GI was not significantly related to UCP excretion [adjusted means (95% CI): 7.27 (6.28, 8.41) vs. 6.51 (5.64, 7.51) vs. 7.94 (6.86, 9.18), P for difference 0.2]. In conclusion, UCP retrospectively measured with a 1-site ELISA remained stable for up to 12 y (from 1990 until 2002). The observed positive relation of UCP with GL appears to result largely from its association with the amount of CHO, whereas dietary GI may be relevant only at higher intake levels. J. Nutr. 136: 1828–1833, 2006. KEY WORDS: urinary C-peptide urine stability children glycemic index glycemic load
Recent epidemiologic studies indicate not only a rising prevalence of childhood overweight and obesity in most industrialized countries (1), but also a considerable number of children and adolescents presenting with signs of the metabolic syndrome or type 2 diabetes mellitus (2,3). Traditionally, dietary recommendations for the prevention and therapy of these abnormalities in children emphasize a reduction of fat intake (4,5). This approach has been criticized because it may lead to a choice of foods with a higher dietary glycemic index (GI),3 i.e., foods yielding higher levels of blood glucose (6). In an experimental study of obese teenage boys, Ludwig et al. (6) found that the consumption of a meal with a high dietary GI was followed by relative hyperglycemia and a high insulin-toglucagon ratio. The downstream effects of this exaggerated response persisted for 2–4 h, even after nutrient absorption had declined, and provoked reactive hypoglycemia followed by counterregulatory hormone secretion and elevated serum free fatty acid concentration 5–6 h after the high-GI meal (6). It was proposed that the repeated occurrence of this metabolic constellation, comparable to a ‘‘state of fasting,’’ promotes excessive
energy intake and impaired b-cell function (7). Should this hypothesis prove to be true, low-GI diets could be an effective dietary means for preventing both overweight and insulin resistance. However, it remains to be determined whether a higher GI or a higher dietary glycemic load (GL: the amount of carbohydrates multiplied by their GI) in the diet of free-living healthy children is also associated with a higher insulin secretion. To address this issue we cross sectionally examined the excretion of C-peptide in 24-h urine samples collected from freeliving healthy 7- or 8-y old-participants of the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study. Urinary C-peptide (UCP) excretion provides a summary measure of insulin secretion over a specific time interval (e.g., 24 h) (8) and may thus be a method of choice when comparing the summary insulin response to different stimuli (8–10). Due to its noninvasiveness, UCP could be of particular interest when repeated measurements of insulin secretion are required in children, e.g., when analyzing individual courses of residual b-cell function in children with type 1 diabetes (11) or long-term patterns of insulin secretion in healthy children. For such retrospective analyses, information on the long-term stability of UCP is required and has been called for explicitly (11,12). However, for biomarkers measured with immunoassays, data from conventional long-term stability studies using split samples are difficult to interpret because the long-term stability of the assay system itself must be guaranteed
1 The DONALD Study is supported by the Ministry of Science and Research of North Rhine-Westphalia, Germany. 2 To whom correspondence should be addressed: E-mail:
[email protected]. 3 Abbreviations used: CHO, carbohydrates; DONALD, Dortmund Nutritional and Anthropometric Longitudinally Designed Study; GI, glycemic index; GL, glycemic load; UCP, urinary C-peptide.
0022-3166/06 $8.00 Ó 2006 American Society for Nutrition. Manuscript received 19 December 2005. Initial review completed 14 February 2006. Revision accepted 20 April 2006. 1828
URINARY C-PEPTIDE AND GLYCEMIC LOAD IN CHILDREN
(11). Alternatively, the concentration of the biomarker measured in similar groups of subjects according to standardized procedures can be compared between different time points (13). The present study, therefore, first determined the long-term stability of UCP frozen at 208C, by comparing UCP levels in 3 similar samples from healthy free-living children aged 7–8 y in 1990, 1996, and 2002 (n ¼ 40 in each period). Equivalent excretions of UCP in these comparable groups would allow us to conclude that UCP is stable for at least 12 y (from 1990 until 2002). In a second step, we examined whether the dietary GI and GL of the total sample (n ¼ 120) were related to the amount of UCP excreted over 24 h.
SUBJECTS AND METHODS Analyses were based on a subsample selected from the DONALD Study, an open cohort study initiated in 1985 to collect detailed data on diet, growth, development, and metabolism between infancy and adulthood (14). The study was approved by the Scientific Committee of the Research Institute of Child Nutrition, and all examinations were performed with parental consent. Annual visits included a medical examination, anthropometric measurements, completion of a weighed 3-d dietary record, and collection of a 24-h urine sample. Body weight was measured to the nearest 0.1 kg using an electronic scale and body height was determined to the nearest 0.1 cm using a digital stadiometer. Micturitions were stored immediately in preservative-free, Extran-cleaned, 1-L plastic containers in home freezers until they could be transported to the Research Institute a few days later. Storage temperature in the home freezer was generally below 188C (minimum: below 128C). At the Research Institute, the urine containers were stored at 208 until they were thawed and stirred so that routine checks could be made and total urine volume determined. Aliquots of 20 mL each were then stored at 228C in the urine bank until further analysis (14). All UCP excretion measurements were carried out simultaneously in 2004 using a solid-phase 1-site polyclonal ELISA (C-Peptid EIA 1293; DRG Instruments), with a detection limit of 0.05 mg/L and an intra- and interassay precision ,7%. For comparative purposes, we subsequently measured the 24-h UCP excretions of 8 women aged 32 6 13 y who consumed a standard diet containing 355 g carbohydrate/d using both a 1- and a 2-site ELISA kit. UCP excretions measured with both systems in freshly collected urine samples were indistinguishable: 15.7 6 3.3 nmol/d [1-site ELISA (DRG)] vs. 15.7 6 4.2 nmol/d (2-site ELISA) using 2 monoclonal antibodies directed against separate antigenic determinants on the C-peptide molecule [Mercodia C-peptide ELISA (specific), Mercodia]. Dietary intake was assessed using weighed 3-d dietary records. Parents of the children weighed and recorded all foods and beverages consumed, as well as leftovers, using electronic food scales (61 g) on 3 consecutive days. Recipes for meals prepared at home were recorded. The packaging of commercial food products was kept. Semiquantitative recording (e.g., number of spoons, scoops) was allowed if weighing was not possible. At the end of the 3-d record period, a dietician visited the family and checked the record for completeness and accuracy. Energy and nutrient intakes were calculated using the Institute’s own nutrient database LEBTAB, which is updated continuously to include all recorded food items. LEBTAB is based on the German standard food composition tables with complementary data from other national food composition tables and data obtained from commercial food products (14). The present analysis was confined to the dietary intake data recorded on the day on which the urine sample was collected. Using published glycemic indices (15), each carbohydrate (CHO)-containing food was assigned a dietary GI according to a standardized procedure (16). In brief, foods were assigned to one of the following: 1) a published GI, 2) the GI of a close match, or 3) the GI calculated from the GI values of the food’s ingredients using recipes available from the in-house database. The CHO content of the food was the principal consideration when matching a particular food with one listed in the tables. Foods containing mainly fat or protein with a CHO content ,5 g/100 g were assigned a GI of 0 (e.g., cold meats).
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All 7- to 8-y-old participants who had collected a 24-h urine sample and had recorded plausible dietary intakes on the same day were eligible for this analysis. Among the 155 children who met these criteria in 1990, 1996, and 2002, 3 samples of 20 boys and 20 girls each were randomly selected for each sampling period (total n ¼ 120). Statistical analyses. The mean daily GI of each subject’s diet was determined by multiplying the CHO content (in g) of each food consumed with the food’s GI (%) and dividing the sum of these products (which corresponds to the GL) by the total daily CHO intake. Earlier studies showed that obese children have higher UCP excretions than normal weight children of the same age, but their UCP/body weight ratios are similar (17). Thus, we always related UCP excretions to body weight (kg) as proposed for children (18). To determine the long-term stability of UCP levels, we first employed the Kruskal-Wallis test, a conventional test for difference, which is based on the null hypothesis that the parameters of interest in the sampling groups are comparable; rejection of this null hypothesis allows the conclusion of difference. In a second step, equivalence was examined by inverse hypothesis testing, i.e., the null hypothesis is that there is an important difference between the groups, and rejection of this null hypothesis allows the conclusion of equivalence (19). Specifically, we employed the formula proposed by Wiens and Iglewicz to test the equivalence of the 3 sampling groups: 0:5
Zmin 5 minij ½2ðjAMi 2AMj j 2 d0 Þ=ðSD2i =ni 1 SD2j =nj Þ ; where Zmin 5 minimum of the pairwise difference test statistics, min 5 minimum, i and j 5 sampling period groups, d0 5 log(1.25), AM 5 arithmetic mean, SD 5 standard deviation, n 5 sample size. Thus, a commonly applied criterion for bioequivalence was used, i.e., for all pairwise comparisons, the ratio of the geometric means had to be between 0.8 and 1.25 (13,19). Equivalence was proven when Zmin exceeded the critical value proposed by Wiens and Iglewicz for d/SEmin, i.e., the ratio of the maximum pairwise difference in sample means (d) to the minimum samples’ standard error (SE). The minimum samples’ standard error was used because the variances differed among the 3 groups. The critical values used herein are also based on the assumption that the ‘‘intermediate’’ group mean value is the arithmetic mean of the lowest and the highest group mean values, i.e., r 5 0.5 (19). Because computation of the minimum of pairwise difference test statistics does not allow conventional adjustment for potential confounders, we computed ‘‘corrected’’ values using the ratio of UCP excretion/potential confounders. Because the distributions of UCP levels and the ratios UCP: potential confounders were skewed, data were log-transformed before statistical analyses. To analyze the association of UCP levels with dietary CHO, GI, or GL, the distributions of CHO, GI and GL were grouped into tertiles. The association was analyzed by least-square regression, calculating geometric mean UCP excretion levels for each tertile adjusted for potential confounders. The adjusted means were the values predicted by the model when the other variables were held at their mean value. To also account for potential confounding by overall energy intake levels, we used the energy partition model, i.e., intakes of fat and protein (in g) were included in the model together with CHO intake. The respective estimates can be interpreted as representing the effect of ‘‘adding’’ CHO or GL, which includes both its energy and nonenergy effect (20), whereas the estimate for GI represents its nonenergy effect only. Tests for differences are based on CHO, GI, or GL being grouped into tertiles, whereas tests for trends consider CHO, GI, or GL as a continuous variable. Differences with P , 0.05 were considered significant. Because analyses indicated no interactions between sex and the relations of the GI or GL to time, UCP excretion, or nutrient intake, data from girls and boys were pooled for analyses. All statistical analyses were carried out using the SAS program, Version 8.2 (21).
RESULTS The characteristics and dietary and urinary variables did not differ among the 3 samples of children from 1990, 1996, and 2002 (Table 1). Overall, mean levels of 24-h UCP excretion,
BUYKEN ET AL.
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TABLE 1 Subject characteristics, and dietary and urinary data in the 3 samples of 7- to 8-y-old healthy children1 Sampling period 1990 (n = 40) Age, y Weight, kg BMI, kg/m2 Carbohydrate intake, g/d GI3 GL, g carbohydrate/d Fiber intake, g/d Protein intake, g/d Fat intake, g/d Energy intake, MJ/d Volume of 24-h urine, L/d 24-h UCP excretion, nmol/d UCP excretion/body weight, pmol/kg UCP excretion/creatinine, nmol/mmol 1 2 3
7.1 24.7 15.6 186 55 109 15 47 63 6.41 590 6.89 297
(7.0, 8.0) (22.5, 26.8) (14.9, 16.3) (170, 233) (51, 57) (90, 127) (12,20) (36, 56) (52, 79) (5.82, 7.42) (507, 768) (5.00, 10.4) (210, 407)
1.83 (1.41, 2.47)
2002 (n = 40)
P for difference2
7.0 (7.0, 8.0) 24.6 (22.7, 28.9) 15.3 (14.7, 16.8) 196 (176, 227) 56 (54, 61) 115 (98, 131) 15 (13,19) 49 (37, 62) 63 (50, 74) 6.48 (5.78, 7.55) 713 (528, 962) 7.48 (5.50, 10.76) 309 (213, 414)
0.3 0.6 0.9 0.8 0.07 0.5 0.09 0.6 0.5 0.9 0.2 0.7 0.7
1.90 (1.52, 2.63)
0.5
1996 (n = 40) 7.0 24.9 15.4 199 57 115 13 49 58 6.52 616 7.17 267
(7.0, 8.0) (23.1, 29.3) (14.8, 16.9) (174, 231) (53, 60) (93, 129) (10,16) (38, 58) (48, 75) (5.28, 7.27) (473, 790) (5.60, 9.95) (225, 382)
1.74 (1.29, 2.24)
Values are medians (Q1, Q3). Kruskal Wallis test for differences among the 3 sampling periods. GI was calculated using the glucose ¼ 100 scale.
GI, and GL of the 7- to 8-y-old healthy children did not differ from those found previously in healthy children (16,18,22). Long-term stability of UCP levels. According to the conservative equivalence test, log-transformed UCP excretions were not equivalent among the 3 sampling periods (Table 2, P . 0.10). Because this could be due to physiological influences, we ‘‘corrected’’ for potential anthropometric and dietary confounders. Simultaneous allowance for body weight and dietary protein intake resulted in equivalence of UCP excretions, i.e., the null hypothesis that the groups differed substantially could be rejected. Similar results were obtained when UCP excretions were corrected simultaneously for body weight and dietary GL (P , 0.05, Zmin 5 1.40). Associations of UCP with dietary CHO, GI and GL. As a result of the findings of the long-term stability analysis, all subsequent models were adjusted for body weight and protein
intake (Table 3, Model 1). Higher UCP excretions were observed in higher tertiles of CHO intake and GL. These associations became (borderline) significant after adjustment for fiber intake (Model 2). Further adjustment for energy intake marginally enhanced these associations (Model 3). For GI, only the suggestion of slightly higher UCP excretions was seen in individuals in the highest dietary GI tertile. Overall, the explained variance in UCP levels was comparable in models for CHO and GL, i.e., the consideration of the GI in addition to the amount of CHO consumed did not improve the explained variance (Table 3). Similar associations between UCP excretions and CHO, GL, or GI occurred when analyses were confined to the 114 normalweight children (data not shown). Furthermore, we also obtained comparable results when examining the association of the UCP:creatinine ratio with dietary CHO, GI, and GL or
TABLE 2 Equivalence test for log-transformed UCP values of 7- to 8-y-old children in 3 sampling periods1 Equivalence test2
Sampling period 1990 (n = 40) Log-transformed UCP excretion Raw, nmol/d Corrected for body weight, pmol/kg Corrected for body weight and protein intake, pmol/(kgg) 1 2
1996 (n = 40) 2002 (n = 40)
Zmin3
P4
1.91 6 0.63 5.60 6 0.65
1.97 6 0.44 5.64 6 0.42
2.04 6 0.40 5.71 6 0.41
0.76 1.00
.0.10 .0.10
1.79 6 0.70
1.80 6 0.46
1.83 6 0.41
1.43
,0.05
Data are log-transformed arithmetic means 6 SD, n ¼ 120. Test for equivalence among the 3 sampling periods using log-transformed values (19). For all pairwise comparison, the ratio of the geometric means is between 0.8 and 1.24. 3 Zmin ¼ minij [ (jAMi AMjj d0)/(SDi2/ni 1 SDj2/nj)0.5], where Zmin ¼ minimum of the pairwise difference test statistics, min ¼ minimum, i and j ¼ sampling period groups, d0 ¼ log(1.25), AM ¼ arithmetic mean, SD ¼ standard deviation, n ¼ sample size. 4 Wiens and Iglewicz provided critical values for a ¼ 0.1, 0.05, 0.025, and 0.010 (19); thus, only estimates of P-values are presented.
URINARY C-PEPTIDE AND GLYCEMIC LOAD IN CHILDREN
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TABLE 3 UCP excretions by tertiles of CHO, GI, and GL in 7- to 8-y-old children1 Tertiles of CHO, GI, or GL 1 CHO [g] UCP [nmol/d] in tertiles of CHO adjusted for Model 1: body weight and protein intake6 Model 2: model 1 1 fiber intake Model 3: model 2 1 energy intake7 GI UCP [nmol/d] in tertiles of GI adjusted for Model 1: body weight and protein intake6 Model 2: model 1 1 fiber intake Model 3: model 2 1 energy intake7 GL UCP [nmol/d] in tertiles of GL adjusted for Model 1: body weight and protein intake6 Model 2: model 1 1 fiber intake Model 3: model 2 1 energy intake7
P for
2
3
162 (118 to ,178)5
197 (178–217)5
256 (218–358)5
6.63 (5.68, 7.73) 6.50 (5.62, 7.54) 6.47 (5.58, 7.50)
7.54 (6.48, 8.78) 7.23 (6.25, 8.37) 7.24 (6.25, 8.38)
7.51 (6.45, 8.75) 7.98 (6.88, 9.26) 8.01 (6.90, 9.30)
56 (54–58)5
61 (.58–71)5
6.53 (5.63, 7.57) 6.51 (5.64, 7.51) 6.40 (5.56, 7.38)
8.21 (7.07, 9.54) 7.94 (6.86, 9.18) 7.82 (6.76, 9.03)
113 (100–123)5
145 (123–203)5
7.86 (6.76, 9.14) 7.82 (6.77, 9.02) 7.82 (6.78, 9.03)
7.51 (6.46, 8.74) 7.76 (6.71, 8.97) 7.80 (6.74, 9.02)
51 (45 to ,54)5 7.00 (6.03, 8.13) 7.27 (6.28, 8.41) 7.50 (6.47, 8.69) 87 (62 to ,100)5 6.35 (5.48, 7.38) 6.19 (5.37, 7.14) 6.15 (5.33, 7.10)
Difference2
Trend3
R2 of model4
0.4 0.2 0.2
0.4 0.06 0.047
8.3% 18.2% 18.8%
0.1 0.2 0.1
0.3 0.8 0.9
8.4% 15.6% 18.8%
0.1 0.04 0.03
0.3 0.09 0.07
8.6% 17.7% 18.3%
1 2 3 4 5 6 7
Values are geometric mean UCP excretion levels (95% CI). Test for difference among the tertiles. Test for trend using CHO, GI, or GL as a continuous variable. Variance in UCP levels explained by the variables included in the respective model, using CHO, GI, or GL as a continuous variable. Values are means (min-max) in tertiles. Adjustment as required according to results from long-term stability analysis (see Table 2). Using the energy partition model (20), i.e., estimates for CHO and GL consider both their energy and non-energy effect; the estimates for GI consider the nonenergy effect only.
when replacing the recorded dietary protein intakes for urinary urea excretion (corrected for creatinine, data not shown), supporting the high quality of the urinary and dietary data.
DISCUSSION Our results show that postprandial insulin secretion in freeliving healthy children, as measured by 24-h UCP excretion, is associated with the dietary GL, but not with the dietary GI. Therefore, the relation of UCP to the dietary GL reflects mainly its association with the amount of dietary CHO. By contrast, the quality of dietary CHO, i.e., the dietary GI, may be relevant only for the 24-h insulin secretion of healthy children consuming a relatively high dietary GI. Although our study is observational rather than experimental, the simultaneous collection of urinary and dietary data used in the DONALD Study allows direct inferences of the effects of dietary exposures on the urinary excretion of biomarkers (23). The findings obtained in the present quasi-experimental study are in line with data from experimental studies in healthy adults (24–26), which reported that higher intakes of dietary CHO yield higher levels of 24-h UCP. Furthermore, in more recent clinical studies, daytime insulin secretions (areas under the curve of insulin measured for 5–24 h) were also related to the amount of dietary CHO in both healthy adolescents (6) and adults (27–30). Our study addressed primarily the effect of dietary CHO on 24-h insulin response; nevertheless, we cannot excluded the possibility that a habitually higher intake of CHO may have contributed to a higher overall b-cell secretory function in some children. However, Sunehag et al. (31) reported a compensatory increase in b-cell secretory function after the consumption of a high-CHO diet for 7 d in overweight adolescents only. Conversely, normal-weight adolescents experienced an
adaptation of carbohydrate oxidation and increased insulin sensitivity in response to a higher CHO intake (32). Experimental studies suggested that the dietary GL may be more closely related to insulin secretion than dietary CHO alone, i.e., that incorporation of the dietary GI enhanced the prediction of the insulin response (33,34). However, in the present study, there was no significant association between the level of the 24-h insulin secretion and the dietary GI of healthy children; the overall proportion of the explained variability in UCP levels was not improved by inclusion of the GI. However, it is noteworthy that ;50% of our participants consumed a diet with an average GI , 55%, i.e., the threshold for a low dietary GI (15). Thus, higher levels of dietary GI may induce higher insulin secretion as indicated by the slightly higher UCP excretion in those children in the highest tertile of GI. Experimental studies in healthy adults that compared low and high GI diets also yielded equivocal data on the association of the dietary GI with 8- to 24-h daytime insulin secretion (28,29,35– 37). Whether a lower dietary GI consumed over the longer term may beneficially affect the insulin sensitivity of healthy children remains to be determined and cannot be addressed in the present analyses because we did not assess the habitual dietary GI or the insulin sensitivity of the children. However, evidence available on this issue for healthy adults from both clinical (28,36,37) and observational studies (38–40) is inconsistent. The associations of UCP with dietary CHO and GL in this study became apparent after adjustment for dietary intakes of fiber and protein, 2 factors that influence insulin secretion (9, 26,41,42). In addition, we could also control for the potential confounding effect of body weight. However, there may be some residual confounding by the children’s physical activity on the day of urine collection, which was not assessed in our study. Children with higher levels of physical activity are more insulin sensitive (43) and are often characterized by a healthier food choice (44), which is in turn associated with the dietary GI (16).
BUYKEN ET AL.
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The use of UCP as a noninvasive measure of insulin secretion (8) was criticized because excretions may vary with different metabolic conditions (11). UCP levels must be interpreted with caution as a quantitative indicator of absolute insulin secretion (8,11,18) as may also be reflected by the relatively low overall proportion of the UCP variation explained by anthropometric and dietary determinants. Nonetheless, UCP is regarded as a useful summary measure of insulin secretion over a specific time interval (e.g., 24 h) in response to a stimulus of interest (8–10), with the advantage of being noninvasive. This study was based on C-peptide excretion in urine samples collected over different time periods, which is frequently the case in longitudinal observational studies. Thus, information on the long-term stability of UCP has been called for explicitly (11). To date, UCP stability has been confirmed only for urine samples collected in adults and stored at 208C over a period of 1 y (45). Our study suggests that C-peptide in the urine of healthy children measured with a 1-site ELISA kit remained stable from 1990 to 2002, i.e., up to 12 y. Equivalence tests comparing samples from similar groups collected at different times (e.g., 1990, 1996, and 2002) may be hampered by time trends in physiological/environmental variables influencing the biomarker (e.g., changes in adiposity and/ or diet). In this study, we could account for bias potentially introduced by 2 major physiological/environmental determinants of UCP excretion because nutritional and anthropometric data, which were collected according to standardized procedures that are monitored regularly for internal validity (14), were available for each sampling period. Bioequivalence became evident after adjustment for body weight and dietary protein intake. We cannot, however, exclude the possibility that the UCP excretions in the present study were influenced by other undetermined factors that differed among the 3 groups (e.g., levels of physical activity). Furthermore, differential degradation of the UCP peptide could have occurred over time, which might have been masked by continued binding of the used polyclonal antibody to potentially emerging UCP degradation fragments. This aspect requires further investigation, e.g., by employing an ELISA which uses 2 (or more) monoclonal antibodies directed against separate antigenic sites of the C-peptide molecule. A preliminary comparison of our one-site ELISA with a 2-site monoclonal ELISA revealed indistinguishable UCP levels, at least in freshly collected 24-h specimens. In conclusion, the statistical equivalence among the UCP excretions of the 3 sampling periods seen after adjustment for the physiologic/environmental determinants of body weight and dietary protein can be regarded as indirect proof of the long-term stability of UCP levels when measured with a 1-site polyclonal ELISA kit. Furthermore, in free-living healthy children, 24-h insulin secretion as reflected by UCP seems to be associated with the dietary GL, i.e., the product of grams CHO and their GI. This relation resulted largely from an association between CHO and UCP, whereas dietary GI may be relevant only at higher intake levels.
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