0 1991 MUNKSGAARD

Human nocturnal blood melatonin and liver acetylation status

Smith JA, Helliwell PS, Isdale A, Astbury C, Padwick DJ, Bird HA. Human nocturnal blood melatonin and liver acetylation status. J. Pineal Res 199 1 : 10: 14-1 7.

Abstract: The human dark phase melatonin concentrations exhibit a wide range of values. In an attempt to explain this variation, we measured 2250-2305 h melatonin levels by radioimmune assay in eleven fast and eleven slow acetylator phenotypes. No statistical difference between the two groups existed, suggesting therefore that such variations are not due to acetylator status. The study revealed a negative relationship between body weight or area and nocturnal melatonin concentration. No correlation was found between dark phase melatonin levels and age, anxiety, depression, or sleep rating.

Normal blood melatonin (MLT) concentration dis- plays a circadian rhythm which normally peaks around 0200 h with a nadir at 1400 h [Arendt et al., 1977; Illnerova et al., 19851. This rhythm is syn- chronous with that of post mortem pineal N-acetyl- transferase (NAT) [Smith et al., 19771, an enzyme that in animals is a rate limiting step in the synthesis of MLT [Illnerova et al., 19831. However, during the dark phase, there is wide variation of plasma MLT concentration at 0200 h consistently reported over many years ranging from 40-250 pg/ml [Smith et al., 1977; Vaughan et al., 1978; Waldhauser et al., 19881. Several factors are known to affect such values. MLT blood and cerebrospinal fluid concentrations in adult subjects have been reported to decrease with age [Brown et al., 1979; Touitou et al., 19811 although in other work no such correlation has been found [Arendt et al., 19821. However, more recently, Waldhauser et al. [ 19881 showed that the negative correlation between serum MLT and age in children and adolescents was probably due to an increase in body weight. In addition, a more moderate decline in serum MLT occurred at older age that was not weight related. A positive correlation with body weight in adults has also been reported [Ferrier et al., 19821. Seasonal variations also exist, with summer and winter plasma MLT concentrations being higher than those occurring in the autumn and spring (Arendt et al.,

John A. Smith,' Philip S. Helliwell,2 Amanda Isdale,2 Carol Astbury,2 David J. Padwick,' Howard A. Bird2 'Pharmaceutical Chemistry, School of Pharmacy, University of Bradford *Clinical Pharmacology Unit (Rheumatism Research), University of Leeds, Royal Bath Hospital, Cornwall Road, Harrogate, U.K.

Key words: Melatonin - polymorphism - slow acetylation - fast acetylation - weight - body area

Dr. J A Smith, Pharmaceutical Chemistry, School of Pharmacy, University of Bradford, Bradford, West Yorkshire, BD7 IDP, U.K.

1 Accepted October 1 , 1990

1979) In addition, melatonin rhythms are reported to be phase advanced in summer [Illnerova et al., 19851.

Other factors such as stress [Vaughan et al., 19791 and activity [Carr et al., 19811, have exhib- ited little or no effect on blood MLT levels. Similarly, some workers show no relationship with sleep [Vaughan et al., 1978; Jimerson et al., 19771 whilst others report sleep patterns do correlate with changes in MLT concentrations in the rat [Mirmiran and Pevet, 19861 and in man [Broadway and Arendt, 19861.

Unlike MLT, which is synthesised in the pineal gland via an acetylation of serotonin by pineal NAT, most biological acetylations are carried out via the non-rhythmic liver NAT. In humans, hepatic NAT is genetically controlled; thus fast and slow acetylation or polymorphism is well documented [Weber 1986; Weber and Hein, 19851. Although there are many differences between liver and pineal NAT [Klein et al., 19851, it is possible that the wide inter-subject variability of human dark phase plasma MLT concentrations may have a genetic basis. In the present work, eleven fast and eleven slow acetylator phenotypes were investigated for 2250-2305 h. plasma MLT concentration. Other parameters such as weight, height, body area, age, anxiety, depression, and sleep ratings were also studied .

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Human melatonin and liver N-acetyltransferase

Subjects, materials and methods Subjects (14 female, age 22-54 yr and 8 male, age 14-53 yr) were recruited from the medical, nursing, and paramedical staff of the Royal Bath Hospital, Harrogate. They were invited to participate by a leaflet explaining the research procedure. Subjects agreeing to participate were given a tablet of 0.5g sulphadimidine that was ingested without food, usually, after an overnight fast. Five to six hours later a 20 ml sample of urine was collected and these samples were frozen at -20°C until analysis.

Subjects randomly selected then returned to pro- vide a blood sample at 2250-2305 h under normal fluorescent lighting. The following variables were recorded: age, sex, height, weight, smoking status, and usual time of retiring and waking. An estimate of the quality of sleep was obtained by asking the subjects to rate their sleep between good, average, and poor. Subjects were also asked to complete the Hospital Anxiety and Depression scale of Zigmond and Snaith [1983]. This rating scale is a self- administered questionnaire designed to provide a quick, reliable estimate of anxiety and depression. The scale has been validated for an out-patient medical population: on each scale scores of 0-7 are regarded as normal, 8-10 as borderline, and 1 1 or more indicative of illness.

Subjects were also advised to avoid alcohol for 24 h prior to the blood sample, and those taking non-steroidal anti-inflammatory drugs and antide- pressant drugs were excluded. The studies were carried out in February 1988.

Ethical Committee approval was obtained from the Leeds General Infirmary Medical Ethics Com- mittee.

Sulphadimidine, acetylsulphadimidine and MLT were purchased from the Sigma Chemical Company. N-[-Zamino-ethyl- 1 -2-3H]-acetyl-5- methoxytryptamine (3HMLT) 30.7 Ci/mmol was supplied by Du Pont (U.K.) Ltd.

Acetylator status was established by the method of Price-Evans [ 19691 by determination of the ratio of acetylsulphadimidine to sulphadimidine in the urine aliquot, using a 65% cut off point to delineate slow from fast acetylators.

Plasma MLT concentration was assessed by the radioimmune assay of Bradbury et al. [1985]. The detection limit was 10 pgiml plasma. The intra assay coefficients of variation (C.V.) (N = 4) were 11% and 10% at 17 and 73 pg/ml respectively and inter assay C.V.s (N = 8) were 6% and 7% at 17 and 73 pg/ml. Recovery of 3HMLT (N = 4) was 73 Ifr 3, 80 rf: 2 and 89 rf: 2% at 20, 40 and 80 pg/ml respectively.

Statistical analysis of the results employed Stu- dent’s t-test to examine differences between the

MLT values in the fast and slow acetylator groups. Linear regression analysis and Pearson’s R correla- tion coefficient were used to examine the relation- ship between MLT values and anthropometric vari- ables such as height, weight, and surface area, using a one tailed test of significance.

Results Table 1 presents the clinical data of the subjects in the study. MLT concentrations measured at 2250- 2305 h in the fast and slow acetylators are shown in Figure 1. Although there was great intersubject variation, no statistical difference was found be- tween the two groups (P = 0.33).

Table 2 demonstrates that there was no signifi- cant difference in dark phase plasma MLT levels, weight, body area, or age between the fast and slow acetylators .

However, a negative correlation was found be- tween weight and dark phase MLT concentration in the total group of subjects (r = -0.36, n = 22, p < 0.05). A negative relationship between body area and dark phase plasma MLT concentration was also established in the total group (r = -0.34, n = 22, P < 0.05).

When plasma MLT was corrected for surface area the differences between the subjects divided according to acetylator status became less evident than when using uncorrected MLT values. No significant relationship between age and 2250- 2305 h plasma MLT concentration was observed over the age ranged examined (14-55 yrs).

There was no significant relationship between anxiety, depression, or sleep rating and dark phase MLT concentration in the total group, nor in any subgroup.

Discussion A wide range of 2300 h plasma MLT concentrations have consistently been reported in man. The levels in this study are consistent with these reports. Although liver NAT exhibits polymorphism [We- ber, 19861, some NAT enzyme from hamster non- liver sources such as spleen, kidney, and pineal have been shown not to be genetically determined [Weber and Hein, 1985; Hein et al., 19861, but polymorphism was evident in NAT taken from intestinal mucosa [Weber and Hein, 19851. The similarities found here between dark phase MLT levels in slow and fast acetylators support the view that pineal NAT does not show polymorphism and thus cannot explain the wide intersubject variabil- ity. Although the single sampling time point (+8 min) occurs during the rising phase of the MLT rhythm, random volunteer selection suggests that it

15

Smith et al.

Table 1. Clinical data of subjects in study ~ ~ ~-

Melatonin Body Area 22.50-23.05h Anxiety Depression

Sex No. Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Female 14 33.8 8.8 64.4 11.1 1.70 0.16 68.6 24.4 1.5 0.52 5.0 2.7 2.0 2.3 Male 8 40.9 13.6 73.1 10.4 1.92 0.11 64.9 15.2 2.0 0.93 6.3 3.7 3.5 3.5

Age (years) Weight (Kg) (m2) pg ml-1 Sleep Rating Rating Rating

140

120

100

80

60

4 0

20

0

A

A

A

A

A

A

0

0 i L B 7 0

0

F A S T S L O W

Fig. I. 22562305 h plasma melatonin concentrations in fast and slow acetylator phenotypes.

is unlikely that any potential differences between fast and slow acetylators would be evened out by taking blood samples from fast acetylators earlier than slow acetylators. More sampling at different time points would provide more information. No difference between fast and slow acetylators was found by Khoory et al. (1980), but in that smaller study, daytime plasma MLT concentrations were measured that are normally low with values ap-

proaching the sensitivity of the assay. However, the negative correlation found between

weight and midnight plasma MLT levels is of interest. Although a positive and negative relation- ship between dark phase MLT and weight has been reported in adults [Ferrier et al., 1982; Touitou et al., 19811, Waldhauser et al. [1988] has shown that the large decrease in plasma MLT from the age of 1-20 yr correlated with an increase in weight, but that the moderate reduction in plasma MLT that was found in the older age groups was due to other factors. The present study is at variance with this view, and may be due to the smaller number of subjects involved. The suggestion by Young et al. [1988] that the negative correlation observed be- tween weight and midnight plasma MLT might be reflected by variations in age is not supported by the results of this study.

A similar negative relationship was found be- tween body area and midnight plasma MLT con- centration in this study, which was accentuated if the wide intersubject range is separated into a high normal, normal, and low normal bands. Although this division is purely arbitrary, such a sub-division may be of some use in subgroup analysis in studies attempting to link abnormal MLT levels with patho- physiological conditions.

No links were observed between MLT concen- tration and either sleep, anxiety, or depression rating. This is not surprising since most subjects were normal on all three scales.

Acknowledgments

We are indebted to Roche Pharmaceuticals for financial support.

Table 2. Fast and slow acetylators compared

Body (m2) Melatonin Age (years) Weight (Kg) SA pg m1-I Sleep Anxiety Depression

No. Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD ~~ ~~

Fast 11 33.7 7.6 66.2 8.6 1.75 0.16 69 28 1.45 0.69 5.72 1.95 3.18 3.66 Slow 11 38.5 13.8 70.6 12.8 1.81 0.21 62 17 1.90 0.70 5.27 4.03 1.91 1.51

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Human melatonin and liver N-acetyltransferase

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