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,3,4,**,5
* Metabolism Unit, Shriners Hospital for Children and Departments of
Surgery and
** Anesthesiology, University of Texas Medical Branch, Galveston, TX 77550
5To whom correspondence should be addressed. E-mail: rwolfe{at}utmb.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • stable isotopes • mass spectrometer • fractional synthetic rate • rabbits
The cellular and protein components of skin are degraded and regenerated continuously (1). This self-renewal of skin is attributed to changes in the rate of division of fibroblasts and keratinocytes, and also to the rate of protein synthesis within those cells. Many skin diseases are directly linked to disruption of the normal function of these types of cells. For example, fibroblasts are involved in the proliferative and synthetic activity required for the formation of hypertrophic scars (1), and psoriasis is associated with a prominent increase in mitotic activity of keratinocytes in the epidermis (2). After skin injury, the stem cells of keratinocytes and fibroblasts are activated to proliferate and differentiate to rebuild the skin barrier (1,2).
Despite the importance of cell proliferation in skin physiology, a method to quantify the rate of in vivo skin DNA synthesis (reflection of cell division) has not been reported. Traditionally, DNA synthesis was measured using either 3H-thymidine or bromodeoxyuridine as the tracer. However, the traditional method is not suitable for in vivo measurement because the tracers are harmful. Further, it determines only the deoxyribonucleoside salvage pathway (Fig. 1). The use of stable isotopes for measurement of DNA synthesis recently overcame the disadvantages of the traditional method (3–6). The stable isotope approach determines the labeling of a purine deoxyribonucleotide, which is a better reflection of DNA synthesis than labeling of a pyrimidine deoxyribonucleotide (4,5). This is because the de novo nucleotide synthesis pathway predominates in the synthesis of deoxyadenosine (dA)6 and deoxyguanosine (dG), and the reuse of dA and dG is minor (4,7–9); the deoxyribose (dR) of dA and dG is derived almost entirely from extracellular glucose, and the reuse of free dR from degraded DNA does not occur (4,7,9). Thus, the de novo purine nucleotide synthesis is regarded as an approximation of total DNA synthesis (4,5).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isotopes. D-[U-13C6]glucose (99% enriched), L-[15N]Gly (98% enriched), and L-[ring-2H5]Phe (98% enriched) were purchased from Cambridge Isotope Laboratories.
Surgical procedures. The rabbits were anesthetized with ketamine and xylazine (10,11). Hair on the neck was removed by a clipper and a lotion hair remover. Using aseptic techniques, Medical Vinyl tubes (Size 3A, Scientific Commodities) with Luer Stub Adapters (Gauge 20, Becton Dickinson) were placed in the left carotid artery and jugular vein via an incision on the neck. The tubes were filled with heparin solution (1000 kU/L) and exited from the back between the ears through a subcutaneous tunnel. Jelco Intermittent injection caps (Johnson & Johnson) were connected to the adapters of the catheters for repeated injections. A skin sample was taken from the incision and immediately frozen in liquid nitrogen. This skin sample was stored at –80°C for later analysis of background enrichment. The incision was closed by layered suturing. Immediately after the surgery, a single dose (50 kU/kg) of antibiotic (Bicillin; Wyeth Laboratories) was injected i.m. When the rabbits awakened from anesthesia, buprenorphrine (0.015 mg/kg) was injected i.m. twice a day for 2 d as an analgesic. Heparin solution (0.45 mL at 1000 kU/L) was injected via the Intermittent injection caps every morning to flush the lines.
Stable isotope infusion. Stable isotope infusion was performed on d 7–9 after surgery. On the day before the isotope infusion, the hair on the back was removed by a clipper and the hair remover. After being washed with water, the prepared skin area was covered with surgical gauze and protected with a rabbit jacket (Harvard Apparatus). Food was removed at 1700 h and the rabbits had free access to water. The infusion study was started at 0800 h on the following day. The catheter in the jugular vein was used for infusion and the catheter in the carotid artery was used for blood withdrawal and measurement of arterial blood pressure on a monitor (mode 78304A, Hewlett Packet). A thermometer probe was taped on the prepared skin for continuous monitoring of skin surface temperature using a digital thermometer.
After a background blood sample was taken from the arterial line, an amino acid solution (100 g/L; Travasol, Baxter Healthcare) was continuously infused for 9 h [2.5 mg/(kg · min); prime 100 mg/kg]. One hour after the start of the amino acid infusion, the infusion of stable isotopes was started. There were 2 groups, an experimental group (n = 6) and a supplemental group (n = 3). In the experimental group, D-[U-13C6]glucose [1.5 µmol/(kg · min); prime 120 µmol/kg], L-[15N]Gly [1.2 µmol/(kg · min); prime 48 µmol/kg], and L-[ring-2H5]Phe [0.2 µmol/(kg · min); prime 8 µmol/kg] were infused to measure FSR of skin DNA and protein. In the supplemental group, only L-[15N]Gly was infused to measure 15N enrichments in aspartate (Asp) and glutamine (Gln). This was to estimate the possible contribution of 15N enrichment in dA by [15N]Gln and/or [15N]Asp through transamination from infused [15N]Gly (12). Arterial blood (1.5 mL each) was drawn every hour during the 8-h isotope infusion period. An additional 1 mL of arterial blood was drawn at 7.5 h for blood gas analysis. When the last blood sample was drawn, a mixture of ketamine and xylazine was injected i.v. for instant anesthesia. A skin sample was taken from the prepared skin area and frozen in liquid nitrogen. This skin sample, along with the background skin sample taken on the day of surgery, was used to measure the enrichments in the free amino acid pools and in the protein-bound and DNA-bound pools. The rabbits were killed under general anesthesia by an i.v. injection of 5 mL saturated KCl.
The mean arterial blood pressure and skin surface temperature were monitored continuously throughout the experiment and recorded every 30 min. During the 9-h experimental period, the rabbits had free access to water and were allowed to move in a restricted area.
Sample analyses
Blood samples. Blood samples were deproteinized with sulfosalicylic acid, and the supernatant was processed for measurement of amino acid enrichments (10). To determine glucose enrichment, plasma was deproteinized with Ba(OH)2 and ZnSO4. The supernatant was processed for measurement of glucose enrichment (13). Blood gas analysis was measured on an Atat Profile 5 Analyzer (NOVA Biomedical).
DNA and protein in whole skin.
The skin samples were kept frozen on dry ice and the panniculus carnosus muscle was removed with a surgical blade. To measure DNA synthesis, 
300 mg of skin was used to extract DNA using a DNA purification kit (Promega). The DNA obtained (50–100 µg) was enzymatically hydrolyzed to nucleosides using a modified procedure described by Chen and Abramson (6). All enzymes used were purchased from Sigma. Briefly, after denaturing the proteins in boiling water, the samples were sequentially incubated with DNase, nuclease P1, and phosphodiesterase at 37°C for 2 h and with NH4HCO3 and alkaline phosphase for another 2 h. The hydrolyzed sample was passed through a 3-mL LC-18 SPE tube (Supelco) to obtain pure dA (14). The pure dA was divided into 2 parts. One (70%) was used for measurement of 15N enrichment in the base of dA; the other (30%) was hydrolyzed into levulinic acid by heating at 100°C for 2 h in 2 mol/L HCl (15) for measurement of 13C enrichment in the dR moiety of dA.
To measure protein synthesis, skin samples of 50 mg each were homogenized in 100 g/L perchloric acid. The supernatant was processed for Gly and Phe enrichment in the free pool in skin. The protein precipitate was processed for L-[ring-5H2]Phe enrichment in the protein-bound pool as described in our earlier studies (10,11).
DNA and protein in dermis. On dry ice, the panniculus carnosus muscle and the epidermis were cut off with a surgical blade. The skin on the back is 3–4 mm in thickness, whereas its epidermal portion is only 75–150 µm thick (16). Therefore, a part of the dermis was cut off along with the epidermis. The stable isotope method, however, does not require complete recovery of the dermis. The obtained dermal tissue, which appears white in contrast to the gray epidermis, was processed for DNA and protein measurement as described for whole skin.
In the supplemental group, the blood and skin samples were processed with special precaution to avoid Gln degradation (13). To this end, an AdVantage Freeze Dryer (VirTis, NY) was used to dry the samples after they passed through the amino acid column.
Measurement of isotopic enrichments. The t-butyldimethylsilyl (TBDMS) derivatives of amino acids were prepared from the supernatant of blood and skin samples (17). Isotopic enrichments were measured on a GC-MS (GC 6890, MSD 5973, Agilent: Hewlett-Packard) with electron impact ionization. Ions were selectively monitored at mass-to-charge (m/z) ratios of 246, 247 for Gly, at m/z ratios of 234 and 239 for Phe, at m/z ratios of 418 and 419 for Asp, and at m/z ratios of 431 and 432 for Gln. Glucose was analyzed as the pentaacetate derivative (13). Glucose enrichment was measured on the GC-MS with electron impact ionization; ions were selectively monitored at m/z ratios of 200.1, 201.1, 202.1, 203.1, 205.1, and 206.1. The amino acids from hydrolysis of protein precipitates were processed for the TBDMS derivatives, and the enrichment of L-[ring-2H5]Phe was measured on a GC-MS (GC 8000 series, MD 800, Fisons Instruments). Ions were selectively measured at m/z ratios of 237 (M + 3) and 239 (M + 5) and converted to the enrichment of M + 5/M + 0 using the standard curve approach (18).
The trimethylsilyl (TMS) derivatization was used for levulinic acid (15) and dA (4). The 13C-levulinic acid and 15N-dA were measured by means of a GC-combustion-isotopic ratio MS (GC-C-IRMS, Finnigan, MAT). The measured 13C enrichment was multiplied by 8/5 to convert to the enrichment of the TMS derivative of levulinic acid because there are 5 carbons in the total of 8 carbons in the molecule that have a chance to be labeled. The measured 15N enrichment was multiplied by 4 to convert to the enrichment of the TMS derivative of dA to account for the fact that only 1 of the 4 nitrogen atoms in the TMS derivative of dA has a chance to be labeled.
The enrichment was expressed as mole % excess except that it was indicated as a tracer/tracee ratio. The enrichment of plasma glucose was corrected for the contribution of the abundance of isotopomers of lower weight to the apparent enrichment of isotopomers with larger weight (19).
Calculations and statistical analysis.
The FSRs of skin protein and DNA were calculated by the tracer incorporation method (13). The general equation is
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where (Et2 –Et1) is the increment of product enrichment from t1 to t2; EP(t2 – t1) is the mean precursor enrichment from t1 to t2. For protein synthesis, the product enrichment is L-[ring-2H5]Phe enrichment in the protein hydrolysate, and the free Phe enrichment in the tissue is used as an approximation of precursor enrichment. For DNA synthesis from the de novo nucleotide synthesis pathway, levulinic acid, which is converted from the dR of dA, is the product and the plasma glucose is the precursor. For DNA synthesis from the de novo base synthesis pathway, the product is the base moiety of dA and the precursor is the free Gly (and Gln) in the tissue (20). DNA FSR from the base salvage pathway = FSR from glucose tracer –FSR from Gly tracer.
Data are expressed as means ± SD. Differences between 2 groups were compared with the nonpaired t test. Differences between whole skin and dermis, between the 2 pathways of DNA synthesis, and between DNA and protein synthesis were compared with the paired t test. The relation between the 2 pathways of DNA synthesis was examined with Pearson’s correlation. A P-value < 0.05 was considered to be significant.
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RESULTS |
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DISCUSSION |
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If the turnover of skin DNA follows the rule that the first DNA molecules to be synthesized are the first to be degraded or lost, then the amount of time that it takes to renew skin DNA is 1/(0.0326/d) = 31 d. Alternatively, if the synthesis and breakdown of skin DNA are random, the amount of time that it takes to renew 50% of skin DNA is (ln 2)/FSR = 0.693/(0.0326/d) = 21 d. Accordingly, it takes 84 d to renew 93.75% of skin DNA. In the epidermis, new keratinocytes are synthesized in the basal layer (or the lowest cell layers of the stratum spinosum), migrate outward, and are then lost by desquamation (2,16,22). This kinetic pattern follows the rule that the first DNA molecules to be synthesized are the first to be degraded or lost. Although the kinetic pattern of dermal fibroblasts is not as clear as epidermal keratinocytes, it is reasonable to estimate that the time span to renew >90% of skin cells is 31–84 d. Based on desquamation rates of the stratum corneum and the consequent obligatory turnover of the keratinocyte layer, the turnover time for human skin epidermis is estimated to be 45–75 d (2). Because of the close FSRs of DNA in whole skin and in dermis (Table 3), it is reasonable to assume that the FSR of epidermal DNA is close to that of whole skin or dermis DNA. Therefore, the FSR of DNA in the skin obtained in the present experiment is considered to agree with current knowledge.
In contrast to the close DNA FSRs between whole skin and dermis, the protein FSR in dermis was less (P < 0.05) than that in whole skin (see Table 3). This was because the epidermal protein has a significantly greater FSR than dermal protein (10). When keratinocytes in the basal layer migrate to the suprabasal layers, they undergo progressive stages of differentiation with synthesis of cytoplasmic proteins (e.g., keratin proteins) and lose the ability to proliferate (2,16). Therefore, it is not surprising that epidermal protein FSR is much greater than epidermal DNA FSR. Such metabolic changes in the epidermis support the notion that cell division and protein synthesis are 2 different cellular processes. This notion is further supported by the finding that there was no significant correlation between DNA and protein FSRs in whole skin and dermis.
The use of the glucose tracer to measure DNA synthesis assumes that the target cells have no significant glyconeogenesis or glycogenolysis, which would dilute the enrichment of glucose-6-phosphate in the intracellular compartment (4). The skin does not have the capacity for gluconeogenesis and has very little glycogen storage (1). Moreover, the epidermal cells use glucose from the circulation rather than intracellular glycogen (1). We measured glucose enrichment in the free pool in skin in 3 rabbits. The glucose enrichment in the skin free pool (10.16 ± 0.27%) was almost identical to that in plasma (10.30 ± 0.36%) both taken at the 8 h time point (unpublished data). Therefore, plasma glucose can be regarded as the precursor for the dR of DNA in the skin.
In the present experiment, we used the skin free amino acids as the precursor for skin protein synthesis and dermal free amino acids as the precursor for dermal protein synthesis as in our previous experiment (10). The free Phe enrichment in the dermis was greater (P < 0.05) than that in whole skin (Table 3). This was because the free Phe enrichment in the epidermis was much smaller than that in dermis (10). Overall tissue free enrichment is used as a surrogate for the true precursor (i.e., aminoacyl-tRNA) enrichment because measurement of aminoacyl-tRNA enrichment in a tissue is technically difficult and results in large variability (23), and tissue free amino acid enrichment is close to the aminoacyl-tRNA enrichment (24). In the present experiment, we infused exogenous amino acids to increase the arterial amino acid concentrations. This might have decreased the enrichment gradient between the extra- and intracellular compartments according to the rationale of the flooding dose technique (25).
In summary, by infusing stable isotope–labeled glucose and glycine, we measured skin DNA synthesis via de novo nucleotide synthesis, de novo base synthesis, and base salvage pathways. The results from the present experiment demonstrated that there is continuous DNA synthesis (i.e., cell division) in the skin, including the dermis. The regulatory mechanism of cell division in the skin could be different from that of protein synthesis. Because cell division and protein synthesis are the 2 principal components of skin metabolism, our method provides a useful tool in the study of not only normal skin but also many skin diseases and wound healing after skin injury.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by The Shriners grants 8630, 8490, and 8790.
3 Current address: JEOL USA, 11 Dearborn Road, Peabody, MA 01960.
4 Current address: U.S. Army Institute of Surgical Research, 3400 Rawley E. Chambers Ave, Fort Sam Houston, TX 78234.
6 Abbreviations used: Asp, aspartate; dA, deoxyadenosine; dG, deoxyguanosine; dR, deoxyribose; FSR, fractional synthetic rate; GC-C-IRMS, gas chromatograph-combustion-mass spectrometer; Gln, glutamine; Gly, glycine; Phe, phenylalanine; TBDMS, t-butyldimethylsilyl; TMS, trimethylsilyl.
Manuscript received 1 May 2004. Initial review completed 24 May 2004. Revision accepted 23 June 2004.
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LITERATURE CITED |
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