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Effect of Lighting Regimen on Simultaneous Diurnal Rhythms of Nucleic and Free Amino Acids in the Liver, Heart, Intestine and Pancreas of the Chick
486
E. F. JANKUS, A. L. GOOD, K. A. JORDAN AND S. K.
REFERENCES Blakely, R. M., 1964. A congestive heart failure condition in turkey breeder males. Poulty Sci. 43 : 1304. Gross, W. B., 1966. Electrocardiographic changes of Escherichia coli-infected birds. Am. J. Vet. Res. 27: 1427-1436. Hamlin, R. L., F. S. Pipers, R. M. Kondrich and C. R. Smith, 1970. QRS component of the orthogonal lead, spatial magnitude and spatial velocity electrocardiograms, and vectorcardiograms of turkeys. J. Electrocardiol. 2: 127-134.
Hunsaker, W. G., 1969. Turkeys have heart attacks too! Canada Agric. J. Canada Dept. Agric, Ottawa, 14: 3-S. Jankus, E. F., and A. L. Good, 1970. Round heart disease in turkeys. Minnesota Veterinarian, 10: 11-12. Jordan, K. S., E. F. Jankus, J. H. Sautter, J. S. Stevens, D. E. Buffington, S. K. Kleven, S. Saxena, A. L. Good, W. A. Junnila and B. S. Pomeroy, 1968. Cardiography of turkeys with round heart involvement. Proc. 21st Annual Conference ACEMB, Vol. 10, Houston, Texas. Magwood, S. E., and D. F. Bray, 1962. Disease condition of turkey poults characterized by enlarged and round hearts. Canad. J. Comp. Med. Vet. Sci. 26:268-272. McCapes, R. H., 1963-64. Report on enlarged heart and ascites. Willow Springs Pedigree Hatches. Purina Company. Unpublished Data. Sautter, J. H., J. A. Newman, S. K. Kleven and C. T. Larsen, 1968. Pathogenesis of the round heart syndrome in turkeys. Avian Dis. 12: 614628. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, p. 67-72.
Effect of Lighting Regimen on Simultaneous Diurnal Rhythms of Nucleic and Free Amino Acids in the Liver, Heart, Intestine and Pancreas of the Chick ROBERT L. SQUIBB
Laboratories
of Disease and Environmental Stress, Rutgers, The State New Brunswick, New Jersey 08903
University,
(Received for publication August 26, 1970)
LTHOUGH the phenomenon of bio- logical rhythms has been known for a long time, it is only in the last few years that a concerted effort has been made by various disciplines to understand them. Many of these biorhythms are definitely circadian (Halberg, 1959) while others, for the present, must be termed oscillations or fluctuations that occur in a 24-hour period with less conformity to clock hours. Research has shown that regardless of classification many of these rhythms can be synchronized or desynchronized by any num-
A
ber of exogenous and/or endogenous agents, a phenomenon which has definite bearing on the interpretation of treament effects. The work reported here was undertaken to determine: 1) the effect of two lighting regimens on diurnal oscillations of nucleic and free amino acids in the liver, heart, intestine and pancreas of normal, rapidly growing chicks; 2) the similarities, if any, of diurnal patterns between tissues; and 3) the extent of "experimental error" if tissue samples were taken only once in a 24-hour
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
control turkeys, 8 days of age and older, have a mean QRS spatial vector directed near the mid-sagittal plane, craniad and dorsad. The RH and healthy 2 day old turkeys have their mean QRS spatial vector directed leftward, caudad and dorsad. The 5 day healthy turkey appears to be in an "electrocardiographic" transitional phase. Utilizing this information, RH was diagnosed and confirmed at autopsy in poults ranging in age from 9 days to 15 months.
SAXENA
487
LIGHTING AND DIURNAL RHYTHMS
period vs. several samplings taken at intervals over the same period. METHODS
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
Day-old White Leghorn cockerels were assigned to two groups of SO birds each and housed in all-wire community cages in identical air-conditioned rooms. Group 1 was put on a constant light (24L) schedule while group 2 received 12 hours of light (12L) each day, from 0700 to 1900 hours. In both rooms light was provided by overhead banks of white fluorescent lights measuring 60 foot candles of light at cage level. Both groups were given the same standard reference diet (Squibb, 1961) and water ad libitum throughout the trial. The animal rooms were entered once a day between 0800 and 0900 hours for care and feeding of the birds. Body weights were taken periodically and monitored against a genetic potential (GP) reference growth curve to ascertain the presence of stress which could confound interpretation of results. Prior to sacrifice all birds had met the GP criteria of maximum growth for the reference diet (Squibb, 1961) and standardized laboratory conditions. Starting at 0800 hours of the day the chicks were 28 days old and continuing at 1500, 2000, 2400 and 0800 hours of the following day 8 birds from each treatment group were weighed and killed. Liver, pancreas, heart, and the small intestine were removed and immediately frozen for later analysis. This procedure always was finished within a 30-minute period and the same personnel participated throughout the trial. All determinations were made on individual tissues except the pancreases which were pooled. Protein and the nucleic acids were analyzed by the modifications of Wannemacher et al. (1965) and seven free amino acids were separated and quantitated by thin-layer chromatography (Squibb, 1963).
RESULTS
Concentrations and total quantities of protein, nucleic and free amino acids in the four tissues, averaged for the 24-hour observation period, are given in Table 1. Lighting regimen had no significant effect on any of the averages. DNA concentrations were lowest in pancreatic tissue and highest in the intestine. RNA was lowest in the heart and highest in the pancreas. The intestine had the lowest amount of protein and pancreas and liver the highest. The liver and intestine had considerably higher levels of free amino acids than the pancreas and heart, the latter being lowest. There was considerable variability in levels of the individual amino acids in the four tissues examined. Patterns of DNA oscillations (Fig. 1) in the liver and heart under 24L were similar and quite different from those in the intestine and pancreas. The 12L regimen tended to invert the liver and heart rhythms, change the intestine pattern altogether, but did not affect the oscillations in the pancreas. Peaks and troughs of RNA in the liver resembled the DNA rhythm but the other RNA curves were irregular under both lighting regimens. In the liver the protein (Fig. 2) fluctuations were similar regardless of lighting schedule; in the intestine and pancreas the patterns were inverted, while in the heart there was no similarity. Total free amino acids showed similar troughs in the intestine but no similarities were evident for the other tissues. Regarding individual free amino acids, patterns for lysine (Fig. 1) were inverted by lighting in the liver, irregular in the heart and pancreas and comparable for the intestine. Liver arginine was inverted by lighting, irregular in the heart and reasonably similar in the intestine and pancreas. Valine patterns (Fig. 2) were irregular in the liver and heart and inverted in the intestine and pancreas. Leucines were also ir-
488
R. L. SQUIBB TABLE 1.—Comparison of the nucleic and f ree amino acid content of several tissues in chicks reared under two lighting regimens Constant Light
Organ weight (g.) Organ wt./body wt. (%) (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet
Total DNA Total RNA Total protein Total FAA's Lys His Arg Asp A Ala Val Leu
(mg.3) (mg.') (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3)
tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue)
Heart
9.1 1 2.5
2.52 0.68
Intestine
— —
Pancreas4
Liver
Heart
1.36 0.37
9.5 2.5
2.49 0.67
1.84 16.8 226 5.74 .91 .46 .65 1.09 1.15 .56 .92
2.06 4.7 193 1.72 .14 .07 .10 .23 .96 .08 .14
2.32 11.9 159 5.88 .77 .52 .82 1.07 1.03 .63 1.04
1.33 36.7 239 3.66 .86 .08 .55 .20 .59 .44 .94
16.7 153 2057 52.3 8.28 4.19 5.92 9.92 10.47 5.10 8.37
5.19 11.8 486 4.33 .35 .18 .25 .58 2.42 .20 .35
— — — — — — — — — —
1.81 49.9 325 4.98 1.17 .11 .75 .27 .80 .60 1.28
1.85 16.6 219 5.56 .88 .47 .63 1.04 1.14 .52 .88
1.99 4.6 180 1.73 .12 .07 .12 .23 .97 .08 .14
4.96 17.6 158 11.5 2081 448 52.8 4.31 8.36 .30 4.47 .17 5.99 .30 9.88 .57 10.83 2.42 4.94 .20 8.36 .35
In
?est' tine
— —
Pancreas4 1.51 0.40
2.34 12.2 153 6.18 .81 .49 .89 1.09 1.09 .73 1.08
1.33 37.1 212 3.14 .62 .12 .45 .25 .54 .30 .86
— — — — — — — — — —
2.01 56.0 320 4.75 .94 .18 .68 .38 .82 .45 1.30
1 All values are averages for 24-hour observation period, 8 chicks/treatment group at each of 5 sampling intervals. 2 Total of 7 free amino acids determined. 3 Total weight of organXmg./g. wet tissue. 4 Pooled samples.
regular in the liver and heart, similar in the intestine and inverted in the pancreas. DISCUSSION Delineation of simultaneous 24-hour fluctuations of several biochemical parameters of protein metabolism in the tissues of young, rapidly growing chicks that met their GP for growth confirms that 1) metabolism is both highly dynamic and irregular, as indicated by the significant diurnal fluctuations of concentrations of tissue constituents; and 2) light is a powerful Zeitgeber. At first glance there seemed to be little similarity in the various diurnal patterns, but close examination revealed that some curves were highly repeatable. For exam-
ple, the 24-hour patterns of liver and heart DNA and RNA have been observed previously in the chick under both 12L and 24L (Squibb, 1968). This repeatability warrants the conclusion that the present findings, despite irregularities, are representative of a true population or normal metabolism and are not reflections of experimental error. In general, at the particular time sampled, concentrations of nucleic and free amino acids in the different tissues changed diurnally and reacted to light in three distinct ways. First, there were inversions in some curves, as illustrated by heart and liver DNA and valine and the leucines in the pancreas. Second, some curves were not affected by light, viz., the DNA and argi-
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
DNA RNA Protein FAA's 2 Lys His Arg Asp A Ala Val Leu
Liver
12 Hour Light (0700-1900 Hours)
LIGHTING AND DIURNAL
LIVER Q O CC UJ Q.
HEART INTEST. PANCR.
LIVER
no
120
100
100
90
80
no
120
100
100
489
RHYTHMS
HEART
INTEST. PANCR.
£
v
UJ
su. UJ CO
LYSINE
x
z < X
o UJ
o cc UJ 0.
90 8
20
J8. 8
80
RNA i
20
•
•
I
X
•
8 8 2 0 8 8 2 0 8
ARGININE •
8
20
•
i
8 8 2 0 8 8
•
•
•
20
•
i
88
20
8
CLOCK HOURS FIG.
1. Effect of lighting on 24-hour patterns of DNA, RNA, lysine and arginine in the liver, heart, intestine and pancreas of the chick. • • constant light; • • 12 hours light daily.
nine patterns in the pancreas and lysine and leucines in the intestine. Third, there were desynchronizations from 12L values and clock hours, as ilhfstrated by DNA in the intestine and RNA, valine, leucines and lysine in the heart. One must assume that metabolic processes within living systems are interrelated. While the data here map some of these simultaneous processes within and between a number of different tissues, as yet they do not provide the basis for understanding the interrelationships of environmental and dietary inputs. Light was incorporated into the experimental design as an exogenous synchronizer calculated to have the least effect on dietary intake and growth rate (Squibb, 1968). The results of this supposedly mild form of stress point out the hazard in making random correlations. For example, un-
der 12L the patterns of lysine in the intestine could have been correlated statistically and biologically with those of dietary intake, the growth process, and liver DNA. However, when the lighting was changed to 24L the lysine patterns in the intestine remained unchanged while those of liver DNA became inverted. In this particular case the difference can be attributed to lighting regimen, but there are other cases where it would be possible for an unknown Zeitgeber input to have caused the same reversal. In general, it becomes evident that for treatments to be both biologically and statistically significant there must be 1) a repeatable pattern for a known biochemical parameter, e.g., liver DNA; and 2) a change in the linearity (slope) of diurnal oscillations (Squibb, 1966, 1968). The data in Table 1 confirm the latter part of
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
3
W
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490
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HEART INTEST. PANCR.
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HEART INTEST. PANCR.
Q
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PROTEIN
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CLOCK HOURS Fio. 2. Effect of lighting on 24-hour patterns of protein, total free amino acids, valine and leucines in the liver, heart, intestine and pancreas of the chick. • • constant light; • • 12 hours light daily.
this thesis since the averages of the five within-day sampling periods indicated that while lighting significantly affected diurnal patterns it did not affect overall 24-hour concentrations. This indicates that in normal animals or those subjected to a mild Zeitgeber there may be a daily balance in turnover or shifting or constituents between tissues. In other words, while the sum total may remain the same over a 24hour period there are specific times during each day when major differences may be observed. Thus the most important information to be gleaned from the data is the elucidation of the way in which periodic changes and Zeitgeber inputs may unknowingly contribute error to what a researcher believes to be a model experiment. Insults to metabolism such as disease and other stressors also may greatly affect 24-hour patterns of periodic reactions
(Squibb, 1968). Averaging values obtained from a series of within-day samplings conducted over a number of days also is not without error because of the possibility of Halberg's serial effects. Tentatively, however, it can be stated that in the chick single samplings obtained between 0800 and 1000 hours will give the closest approximation to those obtained from averages over 24-hour periods. SUMMARY
Simultaneous 24-hour patterns of nucleic and free amino acid values in normal chick liver, heart, intestine and pancreas indicated similarities as well as irregularities, all of which could be affected by lighting regimen. While lighting affected values at specific times of the day, there were no differences in 24-hour averages, indicating periodicity or the dynamism of the living sys-
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
s
'*'
80
; *'
491
LIGHTING AND DIURNAL RHYTHMS
tem as the causative agent. Progressive within-day sampling or single observations conducted at 0800 to 1000 hours over several days or weeks is recommended where treatment effects lack magnitude or are confounded by periodicity. ACKNOWLEDGMENTS
REFERENCES Halberg, F., 1959. Physiologic 24-hour periodicity: General and procedural considerations with reference to the adrenal cycle. Z. Vitamin-Hormon Fermentforsch. 10: 22S-296.
Effect of Lighting Regimen on the Diurnal Distribution of Amino Acids Between Blood Cells and Plasma of Chicks R O B E R T L.
SQUIBB
Laboratories of Disease and Environmental Stress, Rutgers, The State New Brunswick, New Jersey 08903
University,
(Received for publication August 26, 1970)
LOOD amino acids, fueled by dietary sources, are the materia prima for the protein synthetic processes of the body. Consequently, in studies of protein metabolism in various organs and tissues it is important that the interrelationships of the amino acids in the plasma carrier and the formed elements of the bloodstream be clarified (McMenamy et al., 1960). This becomes of even more importance because of known diurnal oscillations or rhythms of blood amino acids (Feigen et al., 1967; Squibb, 1966). The following report concerns 1) the effect of lighting regimen on blood cell/ plasma amino acid ratios; and 2) the effect
B
on these ratios of the addition of four times the requirement of L-lysine to the diet 72 hours prior to the time blood samples were taken. METHODS At time of hatch White Leghorn cockerels were assigned to two equal groups; one was housed in all-wire community cages in an air-conditioned room with constant lighting (24L); the other had identical quarters except that the light was on for 12 hours, from 0700 to 1900 hours daily, the control group (12L). The laboratories were entered once a day from 0800 to 0900 hours by a single caretaker. A standard
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
The work was supported in part by U. S. Public Health Service Grant AM-10673 and the U. S. Army Medical Research and Development Command, Contract No. Da49-193-MD-2694. Marie Utzinger was responsible for the care and management of the chicks and the biochemical analyses.
Squibb, R. L., 1961. Avian disease virus and nutrition relationships. 1. Effect of vitamin A on growth, symptoms, mortality and vitamin A reserves of White Leghorn chicks infected with Newcastle disease virus. Poultry Sci. 40: 425433. Squibb, R. L., 1963. Thin-layer chromatographic separation and quantitative determination of several free amino-acids of avian liver. Nature, 199: 1216. Squibb, R. L., 1966. Diurnal rhythms of tissue components related to protein metabolism in normal and virus infected chicks. J. Nutr. 90: 7 1 75. Squibb, R. L., 1968. Interrelationship of lighting regimen and virus infection to diurnal rhythms in liver components associated with protein metabolism in the chick. J. Nutr. 95: 357-362. Wannemacher, R. W. Jr., W. L. Banks, Jr. and W. H. Wunner, 1965. Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of amino acid incorporation. Anal. Biochem. 11: 320-326.
E. F. JANKUS, A. L. GOOD, K. A. JORDAN AND S. K.
REFERENCES Blakely, R. M., 1964. A congestive heart failure condition in turkey breeder males. Poulty Sci. 43 : 1304. Gross, W. B., 1966. Electrocardiographic changes of Escherichia coli-infected birds. Am. J. Vet. Res. 27: 1427-1436. Hamlin, R. L., F. S. Pipers, R. M. Kondrich and C. R. Smith, 1970. QRS component of the orthogonal lead, spatial magnitude and spatial velocity electrocardiograms, and vectorcardiograms of turkeys. J. Electrocardiol. 2: 127-134.
Hunsaker, W. G., 1969. Turkeys have heart attacks too! Canada Agric. J. Canada Dept. Agric, Ottawa, 14: 3-S. Jankus, E. F., and A. L. Good, 1970. Round heart disease in turkeys. Minnesota Veterinarian, 10: 11-12. Jordan, K. S., E. F. Jankus, J. H. Sautter, J. S. Stevens, D. E. Buffington, S. K. Kleven, S. Saxena, A. L. Good, W. A. Junnila and B. S. Pomeroy, 1968. Cardiography of turkeys with round heart involvement. Proc. 21st Annual Conference ACEMB, Vol. 10, Houston, Texas. Magwood, S. E., and D. F. Bray, 1962. Disease condition of turkey poults characterized by enlarged and round hearts. Canad. J. Comp. Med. Vet. Sci. 26:268-272. McCapes, R. H., 1963-64. Report on enlarged heart and ascites. Willow Springs Pedigree Hatches. Purina Company. Unpublished Data. Sautter, J. H., J. A. Newman, S. K. Kleven and C. T. Larsen, 1968. Pathogenesis of the round heart syndrome in turkeys. Avian Dis. 12: 614628. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, p. 67-72.
Effect of Lighting Regimen on Simultaneous Diurnal Rhythms of Nucleic and Free Amino Acids in the Liver, Heart, Intestine and Pancreas of the Chick ROBERT L. SQUIBB
Laboratories
of Disease and Environmental Stress, Rutgers, The State New Brunswick, New Jersey 08903
University,
(Received for publication August 26, 1970)
LTHOUGH the phenomenon of bio- logical rhythms has been known for a long time, it is only in the last few years that a concerted effort has been made by various disciplines to understand them. Many of these biorhythms are definitely circadian (Halberg, 1959) while others, for the present, must be termed oscillations or fluctuations that occur in a 24-hour period with less conformity to clock hours. Research has shown that regardless of classification many of these rhythms can be synchronized or desynchronized by any num-
A
ber of exogenous and/or endogenous agents, a phenomenon which has definite bearing on the interpretation of treament effects. The work reported here was undertaken to determine: 1) the effect of two lighting regimens on diurnal oscillations of nucleic and free amino acids in the liver, heart, intestine and pancreas of normal, rapidly growing chicks; 2) the similarities, if any, of diurnal patterns between tissues; and 3) the extent of "experimental error" if tissue samples were taken only once in a 24-hour
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
control turkeys, 8 days of age and older, have a mean QRS spatial vector directed near the mid-sagittal plane, craniad and dorsad. The RH and healthy 2 day old turkeys have their mean QRS spatial vector directed leftward, caudad and dorsad. The 5 day healthy turkey appears to be in an "electrocardiographic" transitional phase. Utilizing this information, RH was diagnosed and confirmed at autopsy in poults ranging in age from 9 days to 15 months.
SAXENA
487
LIGHTING AND DIURNAL RHYTHMS
period vs. several samplings taken at intervals over the same period. METHODS
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
Day-old White Leghorn cockerels were assigned to two groups of SO birds each and housed in all-wire community cages in identical air-conditioned rooms. Group 1 was put on a constant light (24L) schedule while group 2 received 12 hours of light (12L) each day, from 0700 to 1900 hours. In both rooms light was provided by overhead banks of white fluorescent lights measuring 60 foot candles of light at cage level. Both groups were given the same standard reference diet (Squibb, 1961) and water ad libitum throughout the trial. The animal rooms were entered once a day between 0800 and 0900 hours for care and feeding of the birds. Body weights were taken periodically and monitored against a genetic potential (GP) reference growth curve to ascertain the presence of stress which could confound interpretation of results. Prior to sacrifice all birds had met the GP criteria of maximum growth for the reference diet (Squibb, 1961) and standardized laboratory conditions. Starting at 0800 hours of the day the chicks were 28 days old and continuing at 1500, 2000, 2400 and 0800 hours of the following day 8 birds from each treatment group were weighed and killed. Liver, pancreas, heart, and the small intestine were removed and immediately frozen for later analysis. This procedure always was finished within a 30-minute period and the same personnel participated throughout the trial. All determinations were made on individual tissues except the pancreases which were pooled. Protein and the nucleic acids were analyzed by the modifications of Wannemacher et al. (1965) and seven free amino acids were separated and quantitated by thin-layer chromatography (Squibb, 1963).
RESULTS
Concentrations and total quantities of protein, nucleic and free amino acids in the four tissues, averaged for the 24-hour observation period, are given in Table 1. Lighting regimen had no significant effect on any of the averages. DNA concentrations were lowest in pancreatic tissue and highest in the intestine. RNA was lowest in the heart and highest in the pancreas. The intestine had the lowest amount of protein and pancreas and liver the highest. The liver and intestine had considerably higher levels of free amino acids than the pancreas and heart, the latter being lowest. There was considerable variability in levels of the individual amino acids in the four tissues examined. Patterns of DNA oscillations (Fig. 1) in the liver and heart under 24L were similar and quite different from those in the intestine and pancreas. The 12L regimen tended to invert the liver and heart rhythms, change the intestine pattern altogether, but did not affect the oscillations in the pancreas. Peaks and troughs of RNA in the liver resembled the DNA rhythm but the other RNA curves were irregular under both lighting regimens. In the liver the protein (Fig. 2) fluctuations were similar regardless of lighting schedule; in the intestine and pancreas the patterns were inverted, while in the heart there was no similarity. Total free amino acids showed similar troughs in the intestine but no similarities were evident for the other tissues. Regarding individual free amino acids, patterns for lysine (Fig. 1) were inverted by lighting in the liver, irregular in the heart and pancreas and comparable for the intestine. Liver arginine was inverted by lighting, irregular in the heart and reasonably similar in the intestine and pancreas. Valine patterns (Fig. 2) were irregular in the liver and heart and inverted in the intestine and pancreas. Leucines were also ir-
488
R. L. SQUIBB TABLE 1.—Comparison of the nucleic and f ree amino acid content of several tissues in chicks reared under two lighting regimens Constant Light
Organ weight (g.) Organ wt./body wt. (%) (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet (mg./g. wet
Total DNA Total RNA Total protein Total FAA's Lys His Arg Asp A Ala Val Leu
(mg.3) (mg.') (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3) (mg.3)
tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue) tissue)
Heart
9.1 1 2.5
2.52 0.68
Intestine
— —
Pancreas4
Liver
Heart
1.36 0.37
9.5 2.5
2.49 0.67
1.84 16.8 226 5.74 .91 .46 .65 1.09 1.15 .56 .92
2.06 4.7 193 1.72 .14 .07 .10 .23 .96 .08 .14
2.32 11.9 159 5.88 .77 .52 .82 1.07 1.03 .63 1.04
1.33 36.7 239 3.66 .86 .08 .55 .20 .59 .44 .94
16.7 153 2057 52.3 8.28 4.19 5.92 9.92 10.47 5.10 8.37
5.19 11.8 486 4.33 .35 .18 .25 .58 2.42 .20 .35
— — — — — — — — — —
1.81 49.9 325 4.98 1.17 .11 .75 .27 .80 .60 1.28
1.85 16.6 219 5.56 .88 .47 .63 1.04 1.14 .52 .88
1.99 4.6 180 1.73 .12 .07 .12 .23 .97 .08 .14
4.96 17.6 158 11.5 2081 448 52.8 4.31 8.36 .30 4.47 .17 5.99 .30 9.88 .57 10.83 2.42 4.94 .20 8.36 .35
In
?est' tine
— —
Pancreas4 1.51 0.40
2.34 12.2 153 6.18 .81 .49 .89 1.09 1.09 .73 1.08
1.33 37.1 212 3.14 .62 .12 .45 .25 .54 .30 .86
— — — — — — — — — —
2.01 56.0 320 4.75 .94 .18 .68 .38 .82 .45 1.30
1 All values are averages for 24-hour observation period, 8 chicks/treatment group at each of 5 sampling intervals. 2 Total of 7 free amino acids determined. 3 Total weight of organXmg./g. wet tissue. 4 Pooled samples.
regular in the liver and heart, similar in the intestine and inverted in the pancreas. DISCUSSION Delineation of simultaneous 24-hour fluctuations of several biochemical parameters of protein metabolism in the tissues of young, rapidly growing chicks that met their GP for growth confirms that 1) metabolism is both highly dynamic and irregular, as indicated by the significant diurnal fluctuations of concentrations of tissue constituents; and 2) light is a powerful Zeitgeber. At first glance there seemed to be little similarity in the various diurnal patterns, but close examination revealed that some curves were highly repeatable. For exam-
ple, the 24-hour patterns of liver and heart DNA and RNA have been observed previously in the chick under both 12L and 24L (Squibb, 1968). This repeatability warrants the conclusion that the present findings, despite irregularities, are representative of a true population or normal metabolism and are not reflections of experimental error. In general, at the particular time sampled, concentrations of nucleic and free amino acids in the different tissues changed diurnally and reacted to light in three distinct ways. First, there were inversions in some curves, as illustrated by heart and liver DNA and valine and the leucines in the pancreas. Second, some curves were not affected by light, viz., the DNA and argi-
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 27, 2015
DNA RNA Protein FAA's 2 Lys His Arg Asp A Ala Val Leu
Liver
12 Hour Light (0700-1900 Hours)
LIGHTING AND DIURNAL
LIVER Q O CC UJ Q.
HEART INTEST. PANCR.
LIVER
no
120
100
100
90
80
no
120
100
100
489
RHYTHMS
HEART
INTEST. PANCR.
£
v
UJ
su. UJ CO
LYSINE
x
z < X
o UJ
o cc UJ 0.
90 8
20
J8. 8
80
RNA i
20
•
•
I
X
•
8 8 2 0 8 8 2 0 8
ARGININE •
8
20
•
i
8 8 2 0 8 8
•
•
•
20
•
i
88
20
8
CLOCK HOURS FIG.
1. Effect of lighting on 24-hour patterns of DNA, RNA, lysine and arginine in the liver, heart, intestine and pancreas of the chick. • • constant light; • • 12 hours light daily.
nine patterns in the pancreas and lysine and leucines in the intestine. Third, there were desynchronizations from 12L values and clock hours, as ilhfstrated by DNA in the intestine and RNA, valine, leucines and lysine in the heart. One must assume that metabolic processes within living systems are interrelated. While the data here map some of these simultaneous processes within and between a number of different tissues, as yet they do not provide the basis for understanding the interrelationships of environmental and dietary inputs. Light was incorporated into the experimental design as an exogenous synchronizer calculated to have the least effect on dietary intake and growth rate (Squibb, 1968). The results of this supposedly mild form of stress point out the hazard in making random correlations. For example, un-
der 12L the patterns of lysine in the intestine could have been correlated statistically and biologically with those of dietary intake, the growth process, and liver DNA. However, when the lighting was changed to 24L the lysine patterns in the intestine remained unchanged while those of liver DNA became inverted. In this particular case the difference can be attributed to lighting regimen, but there are other cases where it would be possible for an unknown Zeitgeber input to have caused the same reversal. In general, it becomes evident that for treatments to be both biologically and statistically significant there must be 1) a repeatable pattern for a known biochemical parameter, e.g., liver DNA; and 2) a change in the linearity (slope) of diurnal oscillations (Squibb, 1966, 1968). The data in Table 1 confirm the latter part of
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CLOCK HOURS Fio. 2. Effect of lighting on 24-hour patterns of protein, total free amino acids, valine and leucines in the liver, heart, intestine and pancreas of the chick. • • constant light; • • 12 hours light daily.
this thesis since the averages of the five within-day sampling periods indicated that while lighting significantly affected diurnal patterns it did not affect overall 24-hour concentrations. This indicates that in normal animals or those subjected to a mild Zeitgeber there may be a daily balance in turnover or shifting or constituents between tissues. In other words, while the sum total may remain the same over a 24hour period there are specific times during each day when major differences may be observed. Thus the most important information to be gleaned from the data is the elucidation of the way in which periodic changes and Zeitgeber inputs may unknowingly contribute error to what a researcher believes to be a model experiment. Insults to metabolism such as disease and other stressors also may greatly affect 24-hour patterns of periodic reactions
(Squibb, 1968). Averaging values obtained from a series of within-day samplings conducted over a number of days also is not without error because of the possibility of Halberg's serial effects. Tentatively, however, it can be stated that in the chick single samplings obtained between 0800 and 1000 hours will give the closest approximation to those obtained from averages over 24-hour periods. SUMMARY
Simultaneous 24-hour patterns of nucleic and free amino acid values in normal chick liver, heart, intestine and pancreas indicated similarities as well as irregularities, all of which could be affected by lighting regimen. While lighting affected values at specific times of the day, there were no differences in 24-hour averages, indicating periodicity or the dynamism of the living sys-
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LIGHTING AND DIURNAL RHYTHMS
tem as the causative agent. Progressive within-day sampling or single observations conducted at 0800 to 1000 hours over several days or weeks is recommended where treatment effects lack magnitude or are confounded by periodicity. ACKNOWLEDGMENTS
REFERENCES Halberg, F., 1959. Physiologic 24-hour periodicity: General and procedural considerations with reference to the adrenal cycle. Z. Vitamin-Hormon Fermentforsch. 10: 22S-296.
Effect of Lighting Regimen on the Diurnal Distribution of Amino Acids Between Blood Cells and Plasma of Chicks R O B E R T L.
SQUIBB
Laboratories of Disease and Environmental Stress, Rutgers, The State New Brunswick, New Jersey 08903
University,
(Received for publication August 26, 1970)
LOOD amino acids, fueled by dietary sources, are the materia prima for the protein synthetic processes of the body. Consequently, in studies of protein metabolism in various organs and tissues it is important that the interrelationships of the amino acids in the plasma carrier and the formed elements of the bloodstream be clarified (McMenamy et al., 1960). This becomes of even more importance because of known diurnal oscillations or rhythms of blood amino acids (Feigen et al., 1967; Squibb, 1966). The following report concerns 1) the effect of lighting regimen on blood cell/ plasma amino acid ratios; and 2) the effect
B
on these ratios of the addition of four times the requirement of L-lysine to the diet 72 hours prior to the time blood samples were taken. METHODS At time of hatch White Leghorn cockerels were assigned to two equal groups; one was housed in all-wire community cages in an air-conditioned room with constant lighting (24L); the other had identical quarters except that the light was on for 12 hours, from 0700 to 1900 hours daily, the control group (12L). The laboratories were entered once a day from 0800 to 0900 hours by a single caretaker. A standard
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The work was supported in part by U. S. Public Health Service Grant AM-10673 and the U. S. Army Medical Research and Development Command, Contract No. Da49-193-MD-2694. Marie Utzinger was responsible for the care and management of the chicks and the biochemical analyses.
Squibb, R. L., 1961. Avian disease virus and nutrition relationships. 1. Effect of vitamin A on growth, symptoms, mortality and vitamin A reserves of White Leghorn chicks infected with Newcastle disease virus. Poultry Sci. 40: 425433. Squibb, R. L., 1963. Thin-layer chromatographic separation and quantitative determination of several free amino-acids of avian liver. Nature, 199: 1216. Squibb, R. L., 1966. Diurnal rhythms of tissue components related to protein metabolism in normal and virus infected chicks. J. Nutr. 90: 7 1 75. Squibb, R. L., 1968. Interrelationship of lighting regimen and virus infection to diurnal rhythms in liver components associated with protein metabolism in the chick. J. Nutr. 95: 357-362. Wannemacher, R. W. Jr., W. L. Banks, Jr. and W. H. Wunner, 1965. Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of amino acid incorporation. Anal. Biochem. 11: 320-326.