The Development of Circadian Rhythms in Human Infants

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Alternative method of urine collection - pads The Newcastle urine collector Uricol pads (Redlands, Newcastle UK) were used as a third alternative method by mothers to collect the night time/day time urine samples. Firstly the infant‟s pubic and perianal area was cleaned and clear of powder, oils and creams. The Uricol pad was accurately positioned over the expected void area cotton side down. The adhesive tape on the back of the pad was removed and the sticky backing attached to the baby nappy. The nappy was then secured. At 10 minute intervals the pad was checked for wetness. As soon as this was confirmed the pad was removed and laid on a flat surface, wet side up. Using the 5ml syringe provided, the tip was placed on the pad at an angle of 45 degrees and 5 ml of urine extracted. The urine was then decanted into a sterile universal container and stored. Parents placed collected urine in universal containers which were placed in household refrigeration facilities. These were then transported the following day to university where they were kept at -20 degrees till processing. 2.4 Estimation of creatinine Creatinine was estimated using the RANDOX Creatinine Colorimetric method, using commercially available reagents (piric acid and sodium hydroxide). The creatinine reacts with the piric acid to form a coloured complex. The amount of complex formed is proportional to the concentration of creatinine (Stockgrand Ltd, Surrey UK). Equal volumes of piric acid and sodium hydroxide are added to make the standard solution. The urine samples are diluted to 1:29 concentration with water. The standard piric acid/sodium hydroxide solution was added to aliquots of the diluted urine samples and quality control high/low creatinine solutions. The samples 70
were read against air at 429 nm after 10 minutes. Concentrations of creatinine were measured in mg ml 1 . 2.5 Estimation of urinary melatonin Values for melatonin estimation were derived by using an assay for a metabolite of melatonin; 6-sulphatoxymelatonin (aMT6s) (Arendt et al., 1985). There is good correlation between urinary 6-sulphatoxymelatonin and urinary/serum melatonin (Bojkowski et al., 1987). The data were log transformed. All values were corrected for renal metabolism using creatinine. Urinary aMT6s assay was conducted using a radioimmunoassay using as 125 I labelled tracer (Stockgrand Ltd, Surrey UK). The diluted urine sample was incubated with a specific sheep antiserum to aMT6s and trace amounts of 125 I-aMT6s was added. The samples are incubated for 15-18 hours at 4 o C. The free and antibody bound fractions of 6-sulphatoxymelatonin are then separated and centrifuged. The non-antibody bound fraction in the pellet is counted in a gamma radiation counter and the 6- sulphatoxymelatonin concentration in the samples determined from the standard dose- response curve that was simultaneously constructed. All samples are processed in duplicate. 71
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The Development of Circadian Rhythm
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Table of Contents LIST OF FIGURES .
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7.1 Analysis of infant sleep measur
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List of Figures Figure 1.1 - Diagra
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List of Tables Table 3.1 - Descript
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1 Introduction Happiness is not a m
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Each of these independently is impo
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There is general agreement, that al
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Summary 1. Development encompasses
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Figure 1.1 - Diagram illustrating c
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A phase shift is used to describe a
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1.4 Ontogeny in Human Infants Well
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1.5.2 Circadian Rhythms in Temperat
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1.5.4 Factors affecting Temperature
Page 29 and 30: 1.6 Melatonin (Reviewed in Cardinal
Page 31 and 32: mechanism by which this occurs is n
Page 33 and 34: It is at this time that infant slee
Page 35 and 36: 1.7 Cortisol Cortisol is a hormone,
Page 37 and 38: 1.7.3 Circadian pattern of Cortisol
Page 39 and 40: In a separate study, Weerth et al u
Page 41 and 42: Summary 1. Cortisol is crucial to a
Page 43 and 44: NREM sleep has 4 stages. It occupie
Page 45 and 46: 1.8.1 Functions of Sleep The functi
Page 47 and 48: 1.8.3 Ontogeny of Sleep in Infants
Page 49 and 50: Sleep can clearly be used as marker
Page 51 and 52: For the purpose of this study, the
Page 53 and 54: Summary 1. Sleep is defined by lack
Page 55 and 56: Overall the SCN generates signals (
Page 57 and 58: Figure 1.2 - The mammalian circadia
Page 59 and 60: 1.9.3 Molecular Components of the C
Page 61 and 62: Figure 1.3 - Mechanisms from SCN to
Page 63 and 64: 1.9.7 Melatonin and Clock Melatonin
Page 65 and 66: The links between the central clock
Page 67 and 68: Chapter 2 Methods
Page 69 and 70: There were strict inclusion and exc
Page 71 and 72: This personal contact on the postna
Page 73 and 74: 2.1.3 Home monitoring visits The fi
Page 75 and 76: 2.1.4 Further monitoring informatio
Page 77 and 78: Core body temperature was taken sim
Page 79: 2.3 Collection of urine samples Pai
Page 83 and 84: allowed the display and comprehensi
Page 85 and 86: 2.8.1 RNA preparation and cDNA synt
Page 87 and 88: In total three methods were trialle
Page 89 and 90: 2.11 Statistical methods For compar
Page 91 and 92: test that each infant‟s height an
Page 93 and 94: Power calculation Sample size: Fift
Page 95 and 96: 3 Results 3.1 Recruited subjects an
Page 97 and 98: predominantly affluent areas of the
Page 99 and 100: Birth history and past medical hist
Page 101 and 102: 3.1.3 Socioeconomic demographics of
Page 103 and 104: her best friend losing her battle w
Page 105 and 106: Sleep environment of infants All mo
Page 107 and 108: Summary 1. This study had higher ra
Page 109 and 110: 4 Results 4.1 Core body temperature
Page 111 and 112: Degrees Celsius 36.70 36.60 36.50 3
Page 113 and 114: 11-13 weeks There is no significant
Page 115 and 116: Core body temperature (degrees Cels
Page 117 and 118: Number of infants 10 8 6 4 2 0 7 8
Page 119 and 120: Mean minimum rectal temperature in
Page 121 and 122: Mean minimum rectal temperature in
Page 123 and 124: Figure 4.7 shows the changes in the
Page 125 and 126: Chapter 5 Result
Page 127 and 128: 4 or 6 hourly samples. Circadian rh
Page 129 and 130: Figure 5.1 - Distribution of mean n
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5.1.2 Emergence of diurnal rhythm f
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Figure 5.3 - Histogram showing dist
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Figure 5.4 - Changes in night time
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Night-day difference in melatonin s
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Summary 1. There is an increase in
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6 Results DESCRIPTION OF THE URINAR
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6.1.2 Emergence of diurnal rhythm f
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Figure 6.3 - Histogram showing dist
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Cortisol values were corrected to a
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6.1.4 Relationship between night an
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Chapter 7 Results
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Mean Length of Sleep/ Wake Bouts Th
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Sleep parameters were recorded as m
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7.3 Development of infant sleep rel
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Figure 7.2 - Changes in infant slee
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Figure 7.4 - Changes in percentage
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Figure 7.5 - Changes in infant frag
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Figure 7.7 - Changes in number of m
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Figure 7.9 - Changes in the percent
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Sleep efficiency (%) 100.0 90.0 80.
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Chapter 8 Results
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Considering only those weeks of mea
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8.2 Description of peripheral gene
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18:30 hours 0.9927; 01:00 ours 1.01
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The weeks after temperature maturat
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Further peripheral gene graphical r
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Raw data points are represented by
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H3f3b/Gap H3f3b/Gap H3f3b/Gap H3f3b
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8.3 Detailed gene expression analys
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Summary 1. H3f3b was selected as ge
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9 Results FACTORS AFFECTING SLEEP A
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Care and infant related factors �
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Table 9.3 - Effect of family occupa
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* Class 7 used as reference group
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Table 9.6 - Effect of measured infa
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Summary: 1. A mature physiological
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10 Results 10.1 Circadian rhythms -
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value of above 0.5 for R squared wa
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Table 10.1 - Temporal relationship
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Chapter 11 Discussion
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� What if any are the factors whi
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11.1 Physiological development - ag
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There are other proven factors whic
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drawbacks of the spread of data - a
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presented in table 6.1 do not have
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separate methods were applied to th
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significant. Sleep latency was cons
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The function of sleep in developmen
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Melatonin secretion was higher on n
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In this study sleeping in the same
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Timing of sleep maturation was diff
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systematic, ordered developmental p
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individual gene plots that provide
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temperature recording devices adapt
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Both melatonin and cortisol rhythms
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Extension of this period of monitor
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Second, we demonstrated the gene of
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Cortisol and melatonin Slope based
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Summary and Future work This study
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� Maximise technique for measurin
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“There is a science of infant car
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References
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Bajanowski, T., Vege, Å., Byard, R
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Chamley, C.A., Carson P, Randall D,
Page 263 and 264:
Frank, M.G., Heller, H.C., 2003. Th
Page 265 and 266:
intrahypothalamic administration of
Page 267 and 268:
Mirmiran, M. & Ariagno, R.L., 2000.
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Rawson, D., Petersen, S.A., Wailoo,
Page 271 and 272:
Stokkan, K.A., Yamazaki, S., Tei, H
Page 273 and 274:
Winter, J.S.D., 1985. The adrenal c
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