PREVENTIVE MEDICINE 13, 670-679 (1984)

Hydroxyproline Excretion in Urine of Smokers and Passive Smokers 1

FRANZ ADLKOFER, *'2 GERHARD SCHERER,* AND W. -D . HELLERY

*Forschungsgesellschaft Rauchen und Gesundheit mbH, Mittelweg 17, D-2000 Hamburg 13, and ?lnstitut fiir Statistik und Mathematische Wirtschaftstheorie, Universitiit Karlsruhe,

Postfach 6380, D-7500 Karlsruhe 1, Federal Republic of Germany

Urinary hydroxyproline excretion was investigated in 125 male cigarette smokers, 194 male pipe and/or cigar smokers, and 24 male nonsmokers. Hydroxyproline excretion was calculated ei ther as hydroxyprol ine/creat in ine ratio or as body surface-standardized amounts of hydroxyproline excreted in urine sampled during day, during night, or over 24 hr. The association of hydroxyproline excretion with smoke uptake variables such as daily cigarette consumption, carboxyhemoglobin, serum cotinine, and nicotine in urine and with self-reported passive smoking exposure in nonsmokers was analyzed. The hydroxyproline/ creatinine ratio was found to be unsuitable as a measure of hydroxyproline excretion since creatinine urine concentrations correlate inversely with smoke uptake in cigarette and pipe/ cigar smokers. The amount of hydroxyproline excreted in 24-hr urine and standardized for body surface was not significantly associated with smoke uptake in pipe/cigar smokers or exposure to passive smoking in nonsmokers. In cigarette smokers the situation appeared similar, although the results were less clear-cut. The data do not favor the premise that measuring urinary hydroxyproline excretion is an accurate method of investigating a lung- damaging effect of smoking, passive smoking, or air pollution. © 1984 Academic Press, Inc.

INTRODUCTION Knowledge of the effects of smoking and passive smoking has been obtained

mainly through epidemiological studies. In order to gain a greater insight into the pathogenesis of smoking-related diseases it is necessary to investigate early ef- fects of smoking on the human organism. Whether or not passive smoking or even active smoking stimulates the degradation of lung collagen and elastin and causes an increased excretion of hydroxyproline in urine is a question that has been discussed over the last few years.

Kasuga and his co-workers (11, 12, 21) have extensively studied urinary hy- droxyproline excretion in relation to exposure to tobacco smoke and polluted air. For practical reasons they used, as is widely accepted, hydroxyproline/creatinine ratios as indicators of hydroxyproline excretion. These authors report a dose- related increase in urinary hydroxyproline excretion in male and female smokers, wives exposed to their husbands' smoking, children exposed to their parents' smoking, and children exposed to automobile exhaust-polluted air (11). They suggest that elevated hydroxyproline/creatinine ratios may be caused by a lung-

1 Presented at the Symposium "Medical Perspectives on Passive Smoking," April 9-12, 1984, Vienna, Austria.

2 To whom requests for reprints should be addressed at: Forschungsgesellschaft Rauchen und Ge- sundheit mbH, Mittelweg 17, D-2000 Hamburg 13, Federal Republic of Germany.

670

0091-7345/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

SYMPOSIUM: MEDICAL PERSPECTIVES ON PASSIVE SMOKING 671

damaging effect of nitrogen dioxide derived from tobacco smoke and air pollution (21). Such an effect of nitrogen dioxide is known from the literature (14).

In several studies involving more than 400 subjects we have investigated the urinary hydroxyproline excretion in cigarette smokers, pipe and/or cigar smokers, and nonsmokers. These studies were originally designed to measure the nicotine uptake in relation to smoking habit. However, they also allowed the examination of urinary hydroxyproline excretion in relation to the subject's smoking habit or exposure to ambient tobacco smoke.

SUBJECTS AND METHODS

Subjects

Male cigarette smokers, pipe and/or cigar smokers, and nonsmokers were re- cruited by newspaper advertisements, by posters in shops, factories, public build- ings, and universities as well as by word of mouth. All subjects filled in a detailed questionnaire related to lifestyle, occupational status, and psychological traits.

Study 1. In the first field study, which lasted from November 1980 to March 1981, 200 cigarette smokers were recruited. Three blood samples were taken from each subject on 3 different days over a period of 3 weeks. The blood samples were taken in our laboratory between 4 and 7 PM. Urine was collected from morning until the time of blood sampling, then continued until the following morning. The samples were categorized as "day" or "night" urine, respectively. Data for day and corresponding consecutive night urines were evaluated as 24- hr urines. To an aliquot of each urine sample 0.1% (v/v) toluene was added. Samples were stored at -20°C until December 1983, when the hydroxyproline and creatinine analyses were performed. Results for day and night urines were evaluated separately. Since not all six urine samples for each subject were avail- able at the time of hydroxyproline determination, it was decided to include the first day or night urine available from each subject for evaluation. Due to phys- iologically raised values in adolescents (10) and, perhaps in some cases, patho- logically elevated values in elderly pipe and cigar smokers (13), it was also decided to base our evaluation only on subjects between ages 21 and 60. Thus 116 day urines, 120 night urines, and 88 24-hr urines from cigarette smokers could be evaluated.

Study 2. In a second study, from May to July 1981, 199 pipe and/or cigar smokers were recruited. From each subject a blood sample was taken between 4 and 7 PM. Before blood sampling, 24-hr urine was collected. Due to availability of urine samples at the time of hydroxyproline and creatinine analyses and after exclusions for age, 154 urine samples from pipe/cigar smokers could be evaluated.

Study 3. In a third study, from March to July 1983, 24 nonsmokers were re- cruited. From each subject a blood sample was taken between 4 and 7 PM. Urine was collected over a period of 24 hr before blood sampling. This group was intended to be the control group for the smokers. One subject had to be excluded due to age younger than 21, lending 23 urine samples from nonsmokers for eval- uation.

672 ADLKOFER, SCHERER, AND HELLER

Analytical Methods

Hydroxyproline and creatinine in urine. Hydroxyproline and creatinine were analyzed 3 with an autoanalyzer (Autoanalyzer Type II, Technicon Co., Ltd.) ac- cording to the methods used by Hosley et al. (9) and Technicon Clinical Methods No. SEZ-0011FC4 (17), respectively.

Nicotine in urine and cotinine in serum. Nicotine in urine and cotinine in serum were determined by gas-liquid chromatography based on the method described by Hengen and Hengen (8). Serum and urine samples were stored at -20°C. All analyses were performed within 6 months after sampling.

Carboxyhemoglobin (COHb). COHb levels were measured with a CO-Oximeter (Instrumentation Laboratories Ltd., Model 182) immediately after taking the blood samples.

Statistical evaluation. Differences between subgroups were tested for signifi- cance using, as appropriate, analysis of variance, the t test, the Wilcoxon test, or the test for linearity.

RESU LTS

Hydroxyproline/Creatinine Ratio

For cigarette smokers, a small, but statistically significant correlation between the hydroxyproline/creatinine ratio and smoke uptake variables such as daily cigarette consumption, COHb, serum cotinine, and nicotine in urine was found, irrespective of whether the analysis was based on urine collected by day or by night (Fig. 1, Tables 1 and 2). In pipe and/or cigar smokers the association between hydroxyproline/creatinine ratio and smoke uptake variables was either not sig- nificant or merely of borderline significance (Table 1). The passive smoke expo- sure is estimated as shown in Table 3. No association between hydroxyproline/ creatinine ratio and passive smoke exposure in nonsmokers was seen, irrespective of whether the analysis was based on the summarizing score (Table 1) or on individual exposures at home or at work (data not shown).

Since changes in the hydroxyproline/creat inine ratio could be caused by changes in either the urinary hydroxyproline or the creatinine concentrations or in both, the association between smoking and urinary creatinine concentrations was checked. Urinary creatinine concentration was found to be negatively cor- related with smoke uptake in cigarette and pipe/cigar smokers (Tables 2 and 4).

Hydroxyproline Excretion Standardized for Body Surface

Hydroxyproline excretion in urine sampled during day, during night, or over 24 hr in relation to different smoking categories for cigarette and pipe/cigar smokers is summarized in Table 5. A significant linear increase in hydroxyproline excretion was found only for night and 24-hr urine of cigarette smokers with

3 All urine samples were analyzed under blind conditions. All available information on the subjects' smoking habits was first deposited with Dr. Dietrich Hoffmann, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla. This was passed on to Professor Kasuga in Japan (Tokai University, School of Medicine, Bohseidai, Isehara 259-11), immediately after the analytical data had been sent to our laboratory.

SYMPOSIUM" MEDICAL PERSPECTIVES ON PASSIVE SMOKING 673

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FIG. 1. Correlation between hydroxyproline (HOP)/creatinine ratio in urine (y axis) and daily cig- arette consumption (x axis) in smokers ages 21-40 years.

increasing serum cotinine levels• All other associations investigated were found to be nonsignificant•

Hydroxyproline excretion in 24-hr urine of nonsmokers was unrelated to their passive smoke exposure, irrespective of whether the analysis was based on their passive smoking scores (Fig. 2) or on individual exposures at home or at the worksite (data not shown)•

Individual hydroxyproline excretions in 24-hr urine samples standardized for body surface for cigarette smokers, pipe and/or cigar smokers, and nonsmokers are shown in Fig• 3. The coefficient of interindividual variation amounted to 48, 39, and 37% for cigarette smokers, pipe/cigar smokers, and nonsmokers, respec- tively. Following up a limited number of subjects (five cigarette smokers and five

TABLE 1 C O E F F I C I E N T S OF C O R R E L A T I O N B E T W E E N H Y D R O X Y P R O L I N E E X C R E T I O N A N D S M O K E U P T A K E

V A R I A B L E S IN S M O K E R S A N D P A S S I V E S M O K E R S a

Smoke uptake variables

Score for Cigarettes Serum Nicotine passive

per day COHb cotinine in urine smoking

Cigarette smokers Day 0.30 (S) 0.39 (S) 0.16 (S) 0.22 (S) - - Night 0.22 (S) 0.17 (S) 0.11 (S) 0.10 (NS) - -

Pipe/cigar smokers - - 0.04 (NS) 0.14 (S) 0.07 (NS) - - Passive smokers . . . . . 0.05 (NS)

a S, significantly different from zero (P < 0.05); NS, not significantly different from zero (P/> 0.05).

674 A D L K O F E R , S C H E R E R , A N D H E L L E R

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nonsmokers) who had been seen three to four times, intraindividual variations of 36 and 50% for nonsmokers and cigarette smokers, respectively, were found.

DISCUSSION

A statistically significant dose -response relationship between cigarette smoking and urinary hydroxyproline/creatinine ratio was found. This is in line with findings by Kasuga et al. (I1). In contrast to Kasuga's results, no sig- nificant association was found between passive smoking and hydroxyproline/cre- atinine ratio. Our findings are not definitive because they are based on an ad- mittedly low number of nonsmokers who reported on their passive smoke ex- posure (Study 3). On the other hand, they should be viewed in conjunction with the results for pipe and/or cigar smokers (Study 2), who may be regarded as highly exposed passive smokers.

However, expressing hydroxyproline excretion by means of the urinary hy- droxyproline/creatinine ratio is only justified if the urinary creatinine concentra- tion is unrelated to smoking. Negative correlations were found between smoke uptake and creatinine concentrations in urine for cigarette and pipe/cigar smokers,

T A B L E 4

COEFFICIENTS OF CORRELATION BETWEEN CREATININE CONCENTRATION IN URINE AND SMOKE

UPTAKE VARIABLES IN SMOKERS OF CIGARETTES AND PIPES AND/OR CIGARS

Smoke uptake variables

Cigarettes per day COHb Serum cotinine Nicotine in urine

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676 A D L K O F E R , S C H E R E R , A N D H E L L E R

T A B L E 5

URINARY HYDROXYPROLINE EXCRETION (--STANDARD DEVIATION) STANDARDIZED FOR BODY

SURFACE IN CIGARETTE AND PIPE/CIGAR SMOKERS CLASSIFIED ACCORDING TO

DIFFERENT SMOKE UPTAKE CATEGORIES

Cigarette smokers (N) Pipe/cigar smokers (N)

" D a y " urine " N i g h t " urine 24-hr urine 24-hr urine Category (mg/mZ/day) (mg/m2/night) (rag/m2/24 hr) (mg/mZ/24 hr)

Cigarettes/day ~<10 6.4 ± 4.4 (20) 10.5 +- 4.5 (19) 16.5 -+ 7.1 (17) 11-20 5.6 ± 3.4 (38) 12.2 -+ 7.3 (40) 17.1 ± 8.9 (27) 21 -30 5.6 ± 3.6 (41) 11.8 -+ 6.9 (41) 18.4 ± 8.9 (31) >30 5.9 ± 4.0 (17) 13.4 +- 5.9 (19) 18.9 ± 8.1 (13)

pa 0.70 0.26 0.35

COHb (%) ~<1.5 5.6 ± 2.6 (37) 10.7 +- 5.8 (35) 16.4 ± 7.3 (29) 1 .6-3 .0 5.9 ± 3.9 (22) 11.8 -+ 7.4 (26) 18.4 ± 10.6 (17) 3 .1-4 .5 4.7 ± 3.0 (20) 13.8 +- 7.1 (21) 19.0 ± 9.8 (13) 4 .6 -6 .0 6.1 -+ 3.6 (16) 11.4 +- 6.8 (18) 16.7 -+ 7.7 (12) >6.0 6.9 -+ 5.3 (21) 13.3 -+ 5.8 (18) 18.8 +- 7.6 (17)

pa 0.27 0.20 0.47

Serum cotinine (ng/ml) ~<75 5.1 ± 2.6 (42) 10.5 _+ 5.9 (39) 15.7 ± 7.4 (34) 76-150 6.8 + 5.1 (23) 10.8 + 5.8 (22) 17.8 + 7.4 (15)

151-225 4.9 ± 2.4 (22) 12.4 ± 7.4 (25) 18.3 + 10.3 (17) 226-300 7.4 _4- 4.8 (12) 12.1 -+ 7.0 (10) 18.7 ± 10.0 (7) >300 6.0 ± 3.9 (17) 15.1 -+ 6.5 (23) 21.0 -+ 8.1 (15)

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23.0 -+ 8.1 (77) 25.4 _+ 11.1 (21) 23.6 -+ 7.4 (16) 28.7 + 13.2 (14) 22.7 ± 9.5 (26)

0.56

Test for linearity.

the association being stronger for cigarette smokers than for pipe/cigar smokers. Thus, the effect of cigarette smoking on hydroxyproline excretion (expressed as hydroxyproline/creatinine ratio) is partly or completely simulated by the dimin-

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SYMPOSIUM: MEDICAL PERSPECTIVES ON PASSIVE SMOKING 677

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ished urinary creatinine concentration in smokers. The cause of this is as yet unclear. Our data suggest, however, that heavy smokers have higher urine vol- umes as compared with light smokers, leading to a lower urinary concentration of creatinine. This, together with the fact that hydroxyproline, in contrast to creatinine, exhibits a high renal reabsorption rate (1, 16), might explain at least part of the association found. On the other hand, some investigators report slightly reduced creatinine serum levels for smokers as compared with non- smokers, which could also cause lower urinary creatinine concentrations (5, 7, 15). This finding has not been confirmed by other authors (2, 4). Whatever the cause for the decreased urinary creatinine concentrations with increased smoke

678 ADLKOFER, SCHERER, AND HELLER

uptake may be, the conclusion must be drawn that hydroxyproline/creatinine ratio is not a suitable parameter to express urinary hydroxyproline excretion in smokers. We, therefore, reevaluated our data using body surface-standardized hydroxyproline excretion in day, night, and 24-hr urine samples. None of the investigated associations were significant except those between serum cotinine and hydroxyproline in night and 24-hr urine samples of cigarette smokers. We do not believe that this result gives any evidence of a collagen- and elastin-degrading effect of tobacco smoke in the smoker's lung, because there are at least three other possibilities that must be considered: (a) this single result may have been obtained by chance; (b) nicotine may enhance the release of growth hormone (20), which seems to be related to an increased urinary hydroxyproline excretion (18); and (c) urinary hydroxyproline excretion may depend on urine volume, which is found to be higher in smokers.

In our view, it is rather unlikely that any assumed damaging effect of smoking on lung collagen or elastin could be demonstrated by measuring hydroxyproline excretion in urine. A large number of factors exert their influence on hydroxy- proline excretion in urine (1). Our finding of high inter- and intraindividual vari- ations support this view. The large amounts of urinary hydroxyproline originating from bone and skin collagen metabolism may prevent the detection of small changes in hydroxyproline derived from lung tissue. Lung collagen and elastin constitute only 1% of total body collagen and elastin (3). For this reason Wendel and Rehpenning (19) do not recommend hydroxyproline measurements for the diagnosis of chronic bronchitis and emphysema. These authors did not find any difference in hydroxyproline excretion between normal subjects and patients with chronic bronchitis and emphysema as long as the heart function of these patients was not decompensated. The same applies for urine desmosine, the crosslinking amino acid unique to elastin. Davies et al. (6) found that urine desmosine is unrelated to cigarette smoking, and attributed their result to high basal urine levels of this amino acid. This suggests that desmosine also cannot be regarded as a useful indicator of lung tissue degradation.

Altogether, our studies do not favor the idea that measuring hydroxyproline excretion in urine is a suitable method for investigating early effects of smoking, passive smoking, and air pollution in man.

ACKNOWLEDGMENTS The authors are indebted to Professor Hitoshi Kasuga and his co-workers at Tokai University for

determining creatinine and hydroxyproline in urine; to Dr. Gisela Henze and Mrs. Claire Hruby for expert assistance in preparing the tables and figures and the manuscript; and to Dr. Dietrich Hoffmann, Naylor Dana Institute, American Health Foundation, Valhalla, New York, who enabled the study to be performed under blind conditions.

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SYMPOSIUM: MEDICAL PERSPECTIVES ON PASSIVE SMOKING 679

3. Buddecke, E. (Ed.). "Grundriss der Biochemie," pp. 471-474. De Gruyter, Berlin/New York, 1973.

4. Chan-Yeung, M., Ferreira, E, Frohlich, J., Schulzer, M., and Tan, E The effects of age, smoking, and alcohol on routine laboratory tests. Amer. J. Clin. Pathol. 75, 302-326 (1981).

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12. Kasuga, H., Matsuki, H., Osaka, F., and Inone, M. The study on the relationship between urinary hydroxyproline and creatinine ratio from the viewpoint of public health. Tokai J. Exp. Med. 4, 343-351 (1979).

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14. Lewis, T. R. Criteria relevant to an occupational health standard for nitrogen dioxide, in "Ni- trogen Oxides and Their Effects on Health" (S. D. Lee, Ed.), pp. 361-375. Ann Arbor Science Publishers, Ann Arbor, Mich., 1980.

15. Mellstroem, D., Rundgren, A., Jagenburg, R., Steen, B., and Svanborg, A. Tobacco smoking, aging, and health among the elderly: A longitudinal population study of 70-year-old men and an age cohort comparison. Age Ageing 11, 45-58 (1982).

16. Sibernagl, S. Renal transport of amino acids. Klin. Wochensehr. 57, 1009-1019 (1979). 17. Technicon Autoanalyzer II: Clinical Method No. SE2-0011 FC4. 18. Wharton, B. A., Brown, G., Rayner, E H. W., Howells, G., and Pennock, C. A. Urinary hy-

droxyproline in children with growth hormone deficiency. Arch. Dis. Child. 49, 159-162 (1974). 19. Wendel, H., and Rehpenning, W. Untersuchungen zur Hydroxyprolinausscheidung im Harn bei

chronischer obstruktiver Bronchitis. Z. gesamte inn. Med. Grenzgeb. 36, 247-249 (1981). 20. Wilkins, J. N., Carlson, H. E., van Vunakis, H., Hill, M. A., Gritz, E., and Jarvik, M. E. Nic-

otine from cigarette smoking increases circulating levels of cortisol, growth hormone, and prolactin in male chronic smokers. Psychopharmacology 78, 305-308 (1982).

21. Yanagisawa, Y., Nishimura, H., Matsuki, H., Osaka, E, Kasuga, H. "Exposure-effect relation for N O 2 with personal exposure and urinary hydroxyproline to creatinine ratio of schoolchil- dren as indicators." Paper presented at the 76th Annual Meeting of the Air Pollution Control Association, Atlanta, Georgia, June 19-24, 1983.