MenstrualCycle

Background: When a human female is born, her ovaries already contain all the immature eggs that will later mature and produce functional eggs during her lifetime. Eggs usually begin to mature between the ages of 12 and 14, when a release of hormones triggers puberty and a young woman reaches sexual maturity. Most commonly, eggs mature every 28 days or so. They usually mature one at a time, in alternating ovaries. This rhythmic maturation of eggs and the other chemical and physical events that accompany the process are called the menstrual cycle.

relation to normal physiological processes. Our present study is concerned with the question of whether urinary polyamine excretion changes during the menstrual cycle. Urinary polyamine excretion does not appear to have been reported in relation to the menstrual cycle.

Urine Specimens
Morning urine specimens were collected daily for about 30 days by 13 healthy women, ages between 20 and 27 years. Precautions were taken to avoid admixture of blood in the urine at the time of menstruation. Voided specimens were centrifuged at 2000 X g (10 mm, 4 #{176}C), frozen, and stored at -25 #{176}C until analysis.
The subjects lived at home during the study and fluid intake, diet, and physical activity were unrestricted. They were not taking an oral contraceptive.
The length of the menstrual cycles ranged from 24 to 36 days. Temperature records were not kept by all subjects, and unequivocable evidence of the occurrence, or absence, of ovulation in each of the cycles studied is not available. Events in the menstrual cycle were dated from the first day on which bleeding occurred.

Polyamine Analysis
Polyamine analysis was done essentially as described by Heby and Andersson (10). Urine (1.00 ml) was hydrolyzed with an equal volume of 12 molt liter HC1 at 110 #{176}C for 14-16 h. After acid hydrolysis, the samples were neutralized with solid Na2CO3 and centrifuged at 2000 X g for 10 mm. A 200-gil aliquot of the supernatant fluid was mixed with 400 il of 1-dimethylamino-naphthalene-5-sulfonyl chloride (30 mg/ml of acetone) and 100 Mlof saturated Na2CO3 solution. The samples were kept in the dark for 14-16 h at room temperature. Excess 1-dimethylamino-naphthalene-5-sulfonyl chloride was eliminated by adding 100 Ml of proline (150 g/liter), which was allowed to react for about 30 mm.
The dansylated polyamines were extracted into 500 Ml of toluene, and the two phases were separated by centrifugation.
A 5-Ml aliquot of each sample was applied on a pre-activated Silica Gel 60 thin-layer chromatographic plate. The plate was developed in chioroform/triethylamine (5/1 by vol) in the dark for about 90 mm. Then it was sprayed with a mixture of triethanolamine/2-propanol (1/4 by vol) to stabilize the fluores- cence. The plate was dried in a desiccator for 1 h in the dark before analysis of the pattern by use of an Aminco-Bowman Spectrophotofluorometer equipped with a TLC-scanner and an XY-recorder. Standard solutions of the polyamines were treated as were the hydrolyzed urine specimens, and 50 to 200 pmol (in 5 tl) of the dansylated derivatives were applied on each plate. The amount of polyammnes in 1 ml of urine was calculated by interpolation from a standard curve, preparedfrom data on the assay of 50, 100, and 200 pmol of each polyamine (n = 16).

Creatinine Determination
We express urinary polyamine concentrations in relation to the excretion of creatinine in the urine. Creatinine was determined according to the procedure described by L#{216}ken (11). In this method, creatinine is reacted with an alkaline picric acid solution to form a colored compound for which there is an absorption maximum at 490 nm. Figures 1A and B show the representative fluctuations in urinary excretion of putrescine, spermidine, and spermine during the menstrual cycle of two healthy individuals.

Results and Discussion
A common feature of all 13 menstrual patternsobtainedwas thatthe combined excretion of all three polyamines was greater during menstruation (Fmgure 2). Sometimes polyamine excretion remained increased during the early follicular phase (Figures 1A  and 2). In addition to the increased polyamine excretion observed during menstruation, all the subjects consistently exhibited one or several mid-cycle peaks in polyamine excretion, roughly coincident with the expected time of ovulation. Frequently, additional peaks were observed in the luteal phase and in the follicular phase as well. However, excretion of polyainines in these phases was not consistently enhanced in all subjects and does not appear to reflect events related to the menstrual cycle. Rather, these peaks may be a function of the diet, because preliminary experiments suggest that the composition of the food plays a role in determining the excretion rate for urinary polyaniines.
In view of the fact that the extracellular polyamine concentration has been shown to increase as a result of cell death (9), it is tempting to speculate that the increase that we consistently observed during menstruation may be related to endometrial necrosis. The fact that significant variations in polyamine excretion were recorded both during individual menstrual cycles and between individuals (Figures 1 and 2) stresses the pointthatsuch possible changes must be taken into account when polyamines are used to monitor therapy of cancer patients. Peak values occurring during normal conditions, particularly during menstruation, even reach values that are regarded as typical for patients with cancer in advanced clinical stages ( Table 1).