Circadian variations of biochemical variables in aspartame treated rats

The following studies were performed on the dangers of aspartame and MSG in relation to the natural circadian rhythm in rats. The studies were designed and performed by the Department of Biochemistry, Faculty of Science Annamalai University, Annamalainagar, Tamil Nadu, India.

Specifically, the researchers observed the effects of aspartame and glucose administration on brain
and plasma levels of large neutral amino acids and brain 5-hydroxy indoles, the effects of intense sweeteners on hunger, food intake and body weight, and the localization of aspartate-like immuno reactivity in the retina of the turtle (Pseudemys scripta).

American critics say these studies bear no effect on the safety of aspartame because they were performed outside the United States. It is true that foreign studies have no influence on American legislation over a product approved by the American FDA, but the most important facts to consider are that these studies were performed under professional laboratory protocols and the results were negative in respect to the safety of consuming aspartame.

Independent American research is sadly falling behind respectable research in other countries. We should be embarrassed that our food science and nutritional research is so lacking. No wonder studies show Americans are sicker than people in European countries.

The Journal of the American Medical Association (May 2006) states Americans spend more on health care than any other industrialized nation, yet trail in life expectancy. Americans spend about $5,200.00 per year on health care while people in the UK spend half that in adjusted dollars.

It's time to pay attention to the "good science" no matter where it is performed opposed to American "corporate science" generating nutritional advice from corporate labs. Americans need to keep up with the times and shift direction toward natural health.

To your health!

Dr. Janet Starr Hull


Pharmaceutical Biology
(Formerly International Journal of Pharmacognosy)
Publisher: Taylor & Francis
Issue: Volume 42, Number 1 / February 2004 Pages: 1 - 7
URL: Linking Options DOI: 10.1080/13880200390500885

Circadian variations of biochemical variables in aspartame treated rats.
P. Rajasekar, P. Subramanian, T. Manivasagam
Department of Biochemistry, Faculty of Science Annamalai University
Annamalainagar Tamil Nadu, India

[ Perumal Subramanian, Thamilarasan Manivasagam ]

Accepted: May 4, 2004
Address correspondence to: P. Subramanian,
Department of Biochemistry,
Annamalai University, Annamalai Nagar - 608 002, Tamil Nadu,
India. E-mail:
DOI: 10.1080/13880200390500885 © 2004 Taylor & Francis Ltd.

In a study of the impact of aspartame on biochemical oscillations, we examined the rhythms of blood glucose, plasma cholesterol, and protein and serum aspartate transaminase (AST) in experimental rats. Our results showed acrophase delays in glucose, total protein and advances in AST rhythms and increased mesor (in AST), amplitude (in cholesterol) and decreased amplitude values (in glucose, AST) in aspartame treated animals. Oral administration of aspartame might lead to increased levels of aspartate in brain which could alter the characteristics of biochemical variables possibly by modulating the transmission in several areas/nuclei in brain including retinohypothalamic tract (RHT) and suprachiasmatic nuclei (SCN).


The biological clock in mammals is located in the suprachiasmatic nuclei (SCN). (Hannibal, 2002). Circadian clocks govern the timing of development, behaviour, physiology, endocrinology and biochemistry, as well as photoperiodic events (Forster et al., 2001). These biological rhythms are adjusted daily (entrained) to the environmental light/dark cycles via retinohypothalamic tract (RHT) (Hannibal, 2002). Many putative neurotransmitters have been identified in the SCN (Vandenpol, 1980).

Aspartate has been reported to be a putative excitatory neurotransmitter in retina (Yagub & Eldred, 1991) in the RHT (Honma et al., 1996) and SCN (Liou et al., 1986; Csaki et al., 2000). This excitatory amino acid is involved in the transmission of light information from retina to SCN via RHT (Takeuchi & Takahashi, 1994). Further, derivatives of aspartate (like N-acetyl aspartyl glutamate) also act as a neurotransmitter in RHT (Hannibal, 2002). Ingestion of L-aspartate into the SCN results in minor phase advances during a subjective day (Devries & Meijer, 1991).

Aspartame (ASP) is a dipeptide artificial sweetener; on oral administration, it is hydrolysed in the gastrointestinal tract to its constituent amino acids, L-phenylalanine and L-asartic acid (Ranney & Oppermann, 1979). Previous studies showed that dietary aspartame could alter the diurnal feeding patterns, meal size, distribution of diurnal pattern of spontaneous motor activity (Torie et al., 1985).

ASP reduced aggressive attack via a serotonergic meachanism (Goerss et al., 2000). The influences of chronic ASP ingestion on brain neuropeptide Y (NPY) concentration, plasma hormone, food intake and body fat in rats have been reported (Beck et al., 2002). The circadian nature of cholesterol synthesis (Jones & Schoeller, 1990) and total protein rhythms in humans and mice (Berezkin et al., 1992) and circadian variation of AST (Coll et al., 1993) were documented.

However, the influences of ASP on biochemical circadian rhythms has not been investigated intensively. In the present study, ASP administration to Wistar rats and its influence on circadian rhythms of blood variables glucose, cholesterol, total protein and AST) were monitored.

Materials and methods


Male Wistar rats (180-190 g) were obtained from the Central Animal House, Annamalai University. They were housed in polypropylene cages and provided food pellets (Agrocorporation Private Limited, Bangalore, India)
as a basal diet during the experiment. Animals were maintained at room tempertature (25 ± 2 °C) and under LD (12 : 12) conditions (Rajakrishnan et al., 1999; Subramanian & Balamurugan, 1999; Subramanian et al., 1998, 2000, 2001). Food and water were available ad libitum throughout the experimental period. Aspartame was obtained from SRL and all other biochemicals used in the study were of analytical grade.

Treatment schedule

The animals were randomized and grouped into experimental and control rats (n = 6 in each group). Group I rats act as control, which received standard food pellets. Group 2 animals were treated orally with aspartame (500 mg/kg body weight) every day (Sharma & Coulombe, 1987; Goerss et al., 2000) throughout the experimental period (11 weeks).

Biochemical oscillations

At the end of the experimental period, a minimal amount of blood (0.75ml) was collected from the orbital sinus using heparinized tubes from normal and aspartame treated groups every 4 hour (00:00, 04:00, 08:00, 12:00, 16:00, 20:00 and 24:00 hour) throughout the 24 hour period (Subrmanian & Balamurugan, 1998; Subramanian et al., 2000). Blood glucose (Fings et al., 1970), plasma cholesterol (Zlatkis et al., 1953), protein (Lowry et al., 1951) and serum AST (Reitman & Frankel, 1957) were estimated after blood collection. At the end of the study both control and ASP treated rats were killed by decapitation and aspartate levels in brain tissues (Pfleiderer, 1969) were measured.

Figure 1. Temporal oscillations of glucose at 4 hour intervals for a period of one day in Wistar rats [control (1A) and aspartame treated (1B)]. Dotted lines represent the raw data and solid lines represent the best fitting cosinor curves (obtained using "cosinorwin" computer software program). Note 6 hour delay of the acrophase in aspartame treated animals (Figure 1B).

Time series analysis

Time series analysis of the oscillation (measurements of acrophase, amplitude and mesor) were done by using "cosinorwin" computer software program. Acrophase is the measure of peak time of the total rhythmic variability in a 24 hour period. mplitude corresponds to half of the total rhythmic variability in a cycle. Mesor (M) is the rhythm adjusted mean. It is equal to the arithmetic mean for equidistant data covering the 24 hour period. The acrophase is expressed in h; mesor and amplitude values are expressed with the same units as the documented variables.

Table 1. Changes in the rhythm characteristics of glucose and cholesterol in control and aspartame treated rats.

Acrophase Amplitude Mesor Biochemical variable Groups (h) (mg/dl) (mg/dl) r-value p-value

Glucose I 00:35 7.3 109.1 0.71dr (p < 0.05)
---------II 7.32 5.3 106.2 -0.34ns (p < 0.50)

Cholesterol I 18:41 8.2 51.9 -0.67dr (p < 0.05)
-----------II 18:49 8.7 51.3 -0.67dr (p < 0.05)

dr -- detectable rhythmicity.
ns -- no significant rhythmicity.

Figure 2. Diurnal rhythms of cholesterol at 4 hour intervals for a period of one day in Wistar rats [control (2A)
and aspartame treated (2B)]. Note modulatory increase in amplitude of the rhythm. No significant changes in acrophase was observed in aspartame treated animals (Figure 2B). Other details as in Figures 1A and 1B.


Aspartate levels in brain tissues (mean ± SD) were increased (4.212 ± 0.41) significantly (p < 0.001) in aspartame treated animals when compared to controls (1.40 ± 0.15).

The endogenous circadian rhythms of glucose showed acrophase at 00:35 hour in normal rats; in the case of aspartame treated animals (group II), maximum levels were found at 7:32 hour (Figure 1A, B). The amplitude, mesor and r-values were decreased significantly in group II when compared with controls (group I). Consinor analysis revealed detectable rhythmicity in normal group and this rhythmicity is affected in aspartame treated group (Table 1).

Cholesterol levels were maximum at 18:41 hour in normal and at 18:49 hour in aspartame treated animals (Figure 2A, 2B). Increased amplitude and decreased mesor values were found in aspartame treated rats than that of normal ones. Detectable cholesterol rhythms were observed in two groups (Table 1).

Table 2. Changes in the rhythm characteristics of total protein and AST in control and aspartame treated rats.

Acrophase Amplitude Mesor Biochemical variable Groups (h) (mg/dl) (mg/dl) r-value p-value

Total protein I 08:48 1.0 5.0 0.02ns (p < 0.5)
-------------II 12:00 0.9 5.2 0.97dr (p < 0.001)

AST I 23:49 10.1 50.9 0.88dr (p < 0.002)
------------II 12:33 4.0 61.9 0.91dr (p < 0.001)

dr -- detectable rhythmicity.
ns -- no significant rhythmicity.

Figure 3. Temporal oscillations of total protein at 4 hour intervals for a period of one day in Wistar rats [control (3A) and aspartame treated (3B)]. Note 4 hour delay of the acrophase in aspartame treated animals (Figure 3B). Other details as in Figures 1A and 1B.

Total protein levels showed the peak value at 8:48 hour and maximum value of aspartame treated animals was observed at 12:00 hour (Figure 3A, 3B). The amplitude and mesor values did not alter significantly in both cases. The rvalue is increased in treated animals than that of normal (Table 2). Analysis of serum AST activity over the 24 hour period revealed that the maximum activity at 23:49 hour in normal and at 12:33 hour in aspartame treated animals (Figure 4A, B). Low amplitude values and increased mesor and r-values were observed in aspartame treated animals, when compared with normal animals (Table 2).


Neurotransmitters involving in regulation of circadian rhythms were normally used to probe the nature of circadian rhythms. Presence of aspartate in RHT (Liou et al., 1986), horizontal, amacrine and ganglion cells, in some photoreceptors and in some unidentified cells in the peripheral retina (Yagub & Eldred, 1991) was reported. The biochemical parameters chosen for this study showed marked fluctuations over a 24 hour period. From this study it can be concluded that light dark cycles are the most effective synchronizers for biochemical circadian rhythms studied in Wistar rats.

In the present study, peak time of glucose was at 3:00 hour in normal and at 7:32 hour in treated animals; this could be attributed to the food intake, digestion, and accumulation of glucose in blood. Rats administered aspartame with showed about 3 hour delays in glucose rhythms. However, no mesor value change was observed in group II when compared with normal rats. Aspartame could not alter glycemia (Ngugen et al., 1998).

Cholesterol levels in rats in the present study were increased at night as reported previously (Ueberberg et al., 1984). The whole body, free and total cholesterol syntheses oscillate periodically and predictably (Jones & Scholler, 1990).

In rats, the rate limiting enzyme (HMG CoA reductase) in the cholesterol synthesis pathway peak its activity at midnight (Mietenen, 1982; Pappu & Illingworth, 1994).

The peak level of transcription of cholesterol-7a-hydroxylse (7aH) gene was reported to occur in the evening. All these factors may contribute to the nocturnal increase of cholesterol.

Figure 4. Temporal variation of AST activity at 4 hour intervals for a period of one day in Wistar rats [control (4A) and aspartame treated (4B)].

Note 11 hour advance of the acrophase in aspartame treated animals (Figure 4B). Other details as in Figures 1A and 1B.

Circadian rhythms in total protein were reported in humans and mice (Touitou et al., 1986; Berezkin et al., 1992). The positive and negative balance between synthesis and degradation of protein might be responsible for the rhythmic phenomenon. In our experiment, AST levels are maximum at 23 hour in control and 11 hour advance in aspartame treated animals. The significant increase in mesor and amplitude of AST rhythm indicated that this may be due to the aspartame metabolites; aspartate might enter the TCA cycle via transamination of aspartate to oxaloacetate (Ranney & Oppermann, 1979).

In the present study, administration of aspsartame increased the brain aspartate levels corroborating the previous results (Moller, 1991; Burns et al., 1991). This increased brain aspartate might favour the transmission of light information to the SCN (Takeuchi & Takahashi, 1994)

Further, aspartame is known to act on NMDA receptors and has some neurological effects seen with glutamate (Disk, 2000; Abdollahi et al., 2001).

Activation of NMDA receptors tends to transmit photic information to the SCN (Mintz et al., 1999). This activation leads to increased production of nitric oxide (NO) (Meller & Gebhart, 1993). Involvement of NO in the transmission of light information to SCN is also suggested (Caillol et al., 2000).

Previous results showed that administration of aspartate into the SCN induced phase shifts in the free running rhythms of hamsters (Smith, 2000).

Hence, we speculate that increased aspartate levels in brain could alter the characteristics of biochemical rhythms studied, possibly by modulating the transmission in several areas/nuclei in brain including RHT and SCN.


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[ Indian J Exp Biol. 2003 Aug; 41(8): 797-804. Circadian clock genes in Drosophila: recent developments. Subramanian P, Balamurugan E, Suthakar G. Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar 608 002, India.

Circadian rhythms provide a temporal framework to living organisms and are established in a majority of eukaryotes and in a few prokaryotes. The molecular mechanisms of circadian clock is constantly being investigated in Drosophila melanogaster. The core of the clock mechanism was described by a transcription-translation feedback loop model involving period (per), timeless (tim), dclock and cycle genes. However, recent research has identified multiple feedback loops controlling rhythm generation and expression. Novel mutations of timeless throw more light on the functions of per and tim products. Analysis of pdf neuropeptide gene (expressed in circadian pacemaker cells in Drosophila), indicate that PDF acts as the principal circadian transmitter and is involved in output pathways. The product of cryptochrome is known to function as a circadian photoreceptor as well as component of the circadian clock. This review focuses on the recent progress in the field of molecular rhythm research in the fruit fly. The gene(s) and the gene product(s) that are involved in the transmission of environmental information to the clock, as well as the timing signals from the clock outward to cellular functions are remain to be determined. Publication Types: Review PMID: 15248475 ]

Posted on June 24, 2006 in Research | Link

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