Tracer enrichment in eye lens connective tissue proteins, CT.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro, (E) Adu-Gly. Male (triangle) and female rat (circle) values are represented as individual enrichment values in the eye lens CT. An outlier on 3.46% enrichment was removed from Wea-Leu (Fig. 7C, n = 9) at day 100. The dashed vertical line indicates the start of the injection period of the tracer. An exponential decay regression curve is marked by a grey line in A (t_1/2: (A) 74.7 days).</p

Tracer enrichment in liver connective tissue proteins, CT.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro, (E) Adu-Gly. Male (triangle) and female rat (circle) values are represented as individual enrichment values (%) in the liver CT. The dashed vertical line indicates the start of the injection period of the tracer. An exponential decay regression curve is marked by a grey line in A—D (t_1/2: (A) 3.7 days, (B) 5.3 days, (C) 2.5 days and (D) 3.5 days).</p

Day 100 tracer enrichment in cytoskeletal/contractile proteins, CYTO.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro. Values are represented as enrichment (%), mean ± 95% CI analyzed in the liver and muscle CYTO, n = 10. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Day 472 tracer enrichment in connective tissue proteins, CT.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro, (E) Adu-Gly. Values are represented as individual enrichments (%), mean ± 95% CI analyzed in the liver, muscle, eye lens and patellar tendon CT, n = 3. Fig. 12A and 12B includes inserts showing the distribution of the enrichment at baseline. The orders of the tissues are the same as the large panels.</p

Day 472 tracer enrichment in cytoskeletal/contractile proteins, CYTO.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro, (E) Adu-Gly. Values are represented as individual enrichments (%), mean ± 95% CI analyzed in the liver and muscle CYTO, n = 3. Fig. 11A includes an insert showing the distribution of the enrichment at baseline. The orders of the tissues are the same as the large panels.</p

Tracer injection protocol.

<p>Lewis rats n = 35 were injected with up to five different tracers (tracer rats). The tracers were injected during the rats’ fetal development (days -10 to -2, with tracer U-¹³C₅-proline, Fet-Pro), after birth (days 5–9, with tracer ring-¹³C₆-phenylalanine, Pup-Phe), at weaning (days 25–32, with tracer 1,2-¹³C₂-leucine, Wea-Leu), at puberty (days 54–58, with tracer ¹⁵N-proline, Pub-Pro) and at adulthood (days 447–451, with tracer 1,2-¹³C₂-glycine, Adu-Gly). The tracers were injected once a day for five consecutive days, except during the fetal development, where injections were done every second day during the second half of the pregnancy of the mother rat. A subset of rats was euthanized on day 1, 12, 32, 45, 61, 63, 69, 81, 100, 454 and 472, after last of the previous injections and on days in between. This was done in order to verify the incorporation of amino acid tracers into the fast turning over proteins. Furthermore, n = 14 rats that didn’t receive any tracer injections (tracer background rats) were euthanized on some of the same days as the tracer rats (day 1, 12, 32, 61, 100 and 472). Only mixed muscle and liver samples are collected from euthanized rats from day 1 and 12 (marked by a hash tag).</p

Tracer enrichment in muscle connective tissue proteins, CT.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro, (E) Adu-Gly. Male (triangle) and female rat (circle) values are represented as individual enrichment values (%) in the muscle CT. The dashed vertical line indicates the start of the injection period of the tracer. An exponential decay regression curve is marked by a grey line in A-D (t_1/2: (A) 3.3 days, (B) 6.8 days, (C) 8.3 days and (D) 23.3 days).</p

Tracer enrichment in liver cytoskeletal proteins, CYTO.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro, (E) Adu-Gly. Male (triangle) and female rat (circle) values are represented as individual enrichment (%) values in the liver CYTO. The dashed vertical line indicates the start of the injection period of the tracer. An exponential decay regression curve is marked by a grey line in A—D (t_1/2: (A) 3.8 days, (B) 4.6 days, (C) 4.6 days and (D) 2.6 days).</p

Body weight, tracer verification and tissue weight.

<p>(A) Mean bodyweight (g) from day 0–472 in male (triangle) and female rats (circle), (B) Tracer enrichment (%) in amino acids collected from serum samples from injected rats at three hours after the first tracer injection for that specific tracer/age (blood sampling +3h), and at one day after the fourth injection and immediately prior to the last/fifth injection (blood sampling +4d) and at a later day: (+22d, +27d, +7d) after the first injection for tracer rats. +3h for Pup-Phe is not measured (X). Values are represented as mean ± 95% CI, (C-D). Relative tissue weight of the soleus muscle, patellar tendon and the eye lens from day 32–472 in male (C) and female (D) rats (% of day 100) (n = 1, except n = 5 for day 100) normalized to day 100.</p

Day 100 tracer enrichment in connective tissue proteins, CT.

<p>(A) Fet-Pro, (B) Pup-Phe, (C) Wea-Leu, (D) Pub-Pro. Values are represented as enrichment (%), mean ± 95% CI analyzed in the CT in liver, muscle, eye lens (one outlier of 3.46% enrichment was removed, Fig. 10C) and patellar tendon, n = 10. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Fig. 10A and 10B includes inserts showing the distribution of the enrichment at baseline. The orders of the tissues are the same as the large panels.</p