QUANTITATIVE HISTOCHEMISTRY OF NICOTINAMIDE ADENINE NUCLEOTIDES IN HUMAN SKIN*

Transcription

1 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Copyright 1970 by The Williams & Wilkins Co. Vol. 55 No. 4 Printed in U.S.A. QUANTITATIVE HISTOCHEMISTRY OF NICOTINAMIDE ADENINE NUCLEOTIDES IN HUMAN SKIN* MICHAEL J. C. IM PH.D.t AND JOHN E. HOOPES M.D.t ABSTRACT The NAD and NADP system was measred in freeze-dried samples (0.3 fj-g) of epidermis hair follicle sebaceos gland sweat gland and dermis by means of enzyme cycling methods. The absolte concentration of ncleotides ranged from to 10- moles in an assay with 1:40000 tisse diltion at the enzyme cycling step. The total NAD was distribted similarly in the taneos organs and its concentration was approximately 1 mfj- mole per mg dry weight. The degree of oxidation of NAD was lowest in sebaceos gland. Less than 40 per cent of the total N AD was in the redced state in the skin. The concentration of total NADP ranged from 100 to 300 fj-p. moles per mg dry weight per cent of which was in the redced form. The concentration of both total N ADP and N ADPH was highest in sebaceos gland; the ratio of N AD to NADP was lowest in sebaceos gland. The higher concentration and higher degree of redction of the NADP system in sebaceos gland coincides with the fact that the activities of NADP-dependent enzymes are higher in sebaceos gland and that this gland is the most active site of lipoo'enesis in skin. The concentration of nicotinamide adenine ncleotides and the degree of their redction increased in proliferating and migrating epithelim dring wond healing. Qantitative histochemical data are available regarding all of the glycolytic enzymes in the kin and its appendages (1). Enzyme analysis provides a valable parameter for the assessment of intermediate metabolic reactions. However thorogh evalation and nderstanding of histochemical activity reqires direct measrement of the metabolic intermediates and cofactors. Techniqes providing these data shold prove more sensitive than analysis of enzyme activities. Measrement of ncleotides in samples as small as moles is possible throgh amplifying the sensitivity of the techniqe by means of enzyme cycling (2). Application of this method to the steady-state measrement of metabolic intermediates cold be accomplished by copling the ncleotide systems with commercially available enzyme preparations (3). The present stdy represents an attempt to adapt the enzyme cycling techniqe to the Received Febrary ; accepted Jne *From the Division of Plastic Srgery Washington University School of Medicine St. Lois Missori t Present address: the Division of Plastic Srgery the Johns Hopkins University School of Medicine Baltimore Maryland investigation of limited skin samples. NAD:t: and NADP and their redced forms have been analyzed in epidermis dermis and dermal appendages. Scce sfl application of this ltramicroanalytic method to the skin will prove sefl in frther defining intermediate metabolic reactions within limited tisse areas: wond healing tmor growth and tisse ltre. MATERIAL AND METHODS Normal abdominal and inginal skin samples obtained from three elderly female patients dring operative procedres were washed in cold rnning tap water for 10 mintes and were frozen immediately in liqid nitrogen. The tisses were sectioned 20 f..t in thickness in a cryostat dried in a vam and stored at -20 C (4). Microscopic dissection of the lyophilized tisses provided 0.3 f..tg samples for analysis. The samples consisted of isolated epidermis exclding stratm cornem hair follicles eccrine and apocrine sweat glands sebaceos glands and dermis. The prity of dissected samples is greater than 90 per cent. One f..tl of cold 0.02 N NaOH containing 1 mm cysteine is added to each tisse sample and the t The abbreviations sed in this paper are as follows: NAD-Nicotinamide adenine dincleotide. N ADH-Redced nicotinamide adenine dincleotide. NADP-Nicotinamide adenine dincleotide phosphate. NADPH-Redced nicotinamide adenine dincleotide phosphate.

2 278 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 60 c: 40. (I) '- 0 :J li:: <] / o ' Toto/ NAD 'o - Total NADP c: (I) '- 0 :J li:: <] 60 O K ~ ~ /.0 Epidermis ( J.l9) Fro. 1. Effect of tisse concentration on vales obtained for total N AD and total N ADP in frozendried hman epidermis. Each point is a mean of triplicate determinations. TABLE I / Ncleotides ( x t0-15 moles) FIG. 2. Standard rve of athentic nicotinamide adenine ncleotide. The concentration of ncleotides is at the enzyme cycling step. Each point is a mean of triplicate determinations. R ecovery of nicotinamide adenine nncleolides added lo samples of f rozen-cl? ied t1:sse Tisse Wt. of tisse N cleotides added Sbstrate measred Fond* Total amont (JLg) ()L~o~m /mg) 0L~o~m /mg ) Epidermis Total NADP I Callated 0.51 NADP NADPH 19G Epidcrmi.R NADPH NADPH Gl Hair follicle ~J ADP 194 Total NADP Hair folliele NADPH NADPH 4-! Epidermis T otal NAD NAD Epidcrmil'l NADH G NADH crin O'land 0.3G 0 NADH NADH * Each figre i a mean of G- 10 analyses. tandard errors are comparable to those given in Tables II and III. tbe are plac d in ice water. Total NAD and N DP is measred in the alkaline sspension withot frther treatment. For the measrement of J DH and N ADPH the sspen ion is heated for 10 mint at 60 C. NAD and N DP a.re nstable in alkali. wherea the redced forms of these ncleotides (NADH NADPH) are qite stable (5). Cycling reagents are prepared as described by Lowry (2) with 50 p.g per ml of glcose-6-phosphate dehydrogenase and 250 IJ.g per ml of glta-

3 NUCLEOTIDES IN SKIN 279 TABLE II Distribtion of total NADP and NADPH in hman skin Specimen* Total NADP NADPH N AD PH/ Total ADP Epidermis I ± 3.3t 66.9 ± II ± ± H air follicle I ± ± II ± ± Sebaceos gland I ± ± II ± ± Eccrine gland I ± ± II ± ± Apocrine gland I ± ± II Dermis I 10.1 ± ± II 6.7 ± ± * Specimen I is from t he inginal skin and II is abdominal. t Concentrations are expressed as J.LJ.L moles/ mg dry weight. Each figre represents a mean of 6-10 determination ± standard error. TABLE III Distribtion of total N AD and N ADH in hman skin Specimen* Total NAD NADH NADH/Total NAD Epidermis I 1040 ± 20t 386 ± II 1122 ± ± Hair follicle I 907 ± ± II 1023 ± ± Sebaceos gl and I 943 ± ± II 917 ± ± Eccrine gland I 884 ± ± II 1377 ± ± Apocrine gland I 1370 ± ± II Derris I II 74 ± ± * Specimen I is from t he inginal skin and specimen II is abdominal. t Concentrations are expressed as J.LJ.L moles/ mg dry weight. Each figre represents a mean of 6-10 determination± standard error. mate dehydrogenase for either NADPH or total NADP and with 20 pg per m1 of lactate dehydrogenase and 200 J.Lg per m1 of gltamate dehydrogenase for NADH or total NAD. T en pl of the appropriate cycling reagent is added to each tbe. NAD and NADP serve as the standards for both the oxidized and redced forms; appropriate blanks are carried throghot the pro-

4 280 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY cedre. The cycling time is 1.5 hors for total NADP NADPH and NADH and one hor for total NAD. The sbseqent reactions after cycling are performed in 3 ml florometer tbes! ~nd the native florescence of NADPH and alkali mdced florescence from N AD are measred in a final volme of 1 ml. Measrement of total NADP and total NAD in variable qantities of lyophyilized epidermis (Fig. 1) proved linear (Fig. 2). Validity of the techniqe was confirmed by recovery experiments in which ncleotides were added to the tisse sample (Table I). RESULTS The concentrations of nicotinamide adenine ncleotides in the skin and its appendages are mmarized in Tables II and III. In general N AD was present in the oxidized form and N ADP was present in the redced form. The content of NAD exceeded that of NADP by a factor of 8-10 in epidermis hair follicle and sweat O'land and by a factor of 4 in sebaceos gland. Total N AD was distribted niformly in the epidermis and dermal appendages (Table III). The sebaceos gland samples exhibited a greater degree of redction ( 45%) than other samples (37%). The percentage of redced N AD in the skin wa higher than in other tisses sch as liver brain and blood. Twenty to 30 per cent of N AD was in redced form in liver and brain and 45 per cent in blood (2). Total N ADP also was distribted niformly except for a 2-fold increase in the sebaceos gland samples. The degree of redction was high in epidermis sebaceos glands and hair follicl and low in sweat glands and dermi (Table II). The NADPH/ total NADP ratio wa 0.62 in epidermis 0.72 in sebaceos gland and 0.6 in hair follicle; and 0.53 in apocrine sweat gland and dermis and 0.34 in eccrme sweat gland. The ratio of NADPH to total NADP was 0.80 in liver 0.75 in brain and 0.45 in blood (2). Similar measrements were ndertaken in the regenerating epithelim three days following experimental wonding in ginea pigs to frther investigate the dynamics of the N AD and NADP systems. A gradient for NADH was observed between normal proliferating and migrating epithelim with a dramatic increase being noted in the NADH/total NAD ratio in migrating epithelim (Table IV). The repairing epithelim contained an increased concentration of NADPH (Table IV); the NADPH/ total NADP ratio was increased in both marginal proliferating epithelim and migrating epithelim. DISCUSSION The NAD/ NADP ratio is low in sebaceos glands as compared to other taneos organs and this may indicate a predominance of the NADP system in sebaceos glands. The finding of a higher concentration of the NADP system with a high concentration of its redced form in sebaceos glands is not srprising. Sebaceos glands have higher activities of NADPH prodcing enzymes sch as glcose 6-phosphate 6-phosphoglconate and isocitrate dehydrogenase (7 8). Abndance of NADPH in tisse is essential for synthetic processes and sebaceos glands are the most active sites of lipogenesis in the skin. The balance stdy of cytoplasmic nicotinamide adenine ncleotides indicates an excess spply of NADPH in hair follicles especially in growing follicles (9). The low nicotinamide adenine ncleotide content of dermis is in accord with the general inactivity of a variety of enzymes. Previos investigations based on analysis of TABLE IV Changes of nicotinamide adenine ncleotides in the regenerating epithelim of 3-day old wond on the back skin of ginea pig Concentration ar expressed as JJ.JJ. moles per mg dry weight tisse. Each figre represents a mean of 6 determinations ± tandard error. Epithelim Total NADP NADPH AD PH/total NADH/ total NADP NAD Total NAD NADH Normal 61.5 ± ± ± ± Marginal 9.5 ± ± ± ± Migratin ± ± ± ± 22 I 0.69

5 :NUCLEOTIDES IN SKIN 281 enzyme activity have demonstrated the increased enzyme activities of marginal proliferating and migrating epithelim (6). A prominent increase in glcose-6-phosphate dehydrogenase and some glycolytic enzyme activities has been observed dring the first three days of wond healing. Isotopic stdy on the over-all metabolic reaction has demonstrated that the contribtion of the pentose phosphate cycle to glcose metabolism and the rate of the net formation of cytoplasmic NADH and NADPH increase in 3 day wonds (10). The increased concentrations of redced NAD and NADP in regenerating epithelim correlate well with the data derived from enzyme analyses. The significance of the present stdy lies in demonstrating that the enzyme cycling techniqe is applicable to the skin and its appendages and in demonstrating that this techniqe can be sed for qantitative histochemical measrement of metabolic intermediates in small amonts of tisse. Histochemical distribtion of certain metabolites is being elcidated in rrent investigations i.e. glycogen is present in high concentration in hair follicles and eccrine sweat glands and ATP is present in high concentration in epidermis and eccrine sweat glands. REFERENCES 1. Adachi K.: Enzyme activities in mammalian. pigment cells p. 223 Advances in Biology of Skin Vol. 8. Eds. Montagna W. and H. F. Pergamon Press Oxford Lowry 0. H. Passonnea J. V. Schlz D. W. and Rock M. K.: The measrement of pyridine ncleotides by enzymatic cycling. J. Biol. Chern. 236: Gatfield P. D. Lowry 0. H. Schlz D. W. and Passannea J. V.: Regional energy reserves in mose brain and changes with ischemia and anesthesia. J. Nerochem. 13: Lowry 0. H.: Qantitative histochemistry of brain: histological sampling. J. Histochem. Cytochem. 1: Lowry 0. H. Passannea J. V. and Rock M. K.: The stability of pyridine ncleotides. J. Biol. Chern. 236 : Im M. J. C. and Hoopes J. E.: Enzyme activities in the repairing epithelim dring wond healing. J. Srg. Res. 10: H ershey F. B. Lewis C. Mrphy J. and Schiff T.: Qantitative histochemistry of hman skin. J. Histochem. Cytochem. 8: Hershey F. B.: Hormone and enzyme mechanisms in sebaceos gland excretions. J. Invest. D erm. 32: Adachi K. and Uno H.: Some metabolic profiles of hman hair follicles Advances in Biology of Skin Vol. 9. Eds. Montagna W. and Dobson R. Pergamon Press Oxford Im M. J. C. and Hoopes J. E.: Energy metabolism in healing skin wonds. In preparation.