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Cats Require More Dietary Phenylalanine or Tyrosine for Melanin Deposition in Hair than for Maximal Growth


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In 1986, the NRC recommended a dietary concentration of 4.0 g/kg of phenylalanine and 8.5 g/kg of total aromatic amino acids for growing kittens on the basis of maximal growth rate and nitrogen balance. Black hair-coated cats were given purified diets containing the following phenylalanine + tyrosine (Phe + Tyr) concentrations (g/kg diet): 4 + 2, 4 + 4, 4 + 6, 4 + 8, 10 + 0, 10 + 2, 10 + 4, 10 + 6, 10 + 8, 10 + 10, 24 + 0 for at least 6 mo. All other amino acids were present at about twice the requirements. Total melanin and the oxidation product of eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA) were measured in hair. There was a significant linear relationship between the concentrations of tyrosine in plasma and PTCA in hair. The relationship between PTCA concentration in hair and Phe + Tyr in the diet had a point of inflection at approximately 16 g/kg Phe + Tyr. Cats fed diets with <16 g Phe + Tyr developed "red hair." We confirmed the anecdotal reports that the black hair of cats can change from black to reddish brown. An aromatic amino acid concentration > or =18 g/kg is recommended for the prevention of visually discernible red hair in black-coated cats. Dietary concentrations >18 g total aromatic amino acids/kg diet promote a greater ratio of PTCA:total melanin in hair. We are unaware of a secondary nutrient requirement being so much greater than the requirement for growth.
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Nutrient Requirements
Cats Require More Dietary Phenylalanine or Tyrosine for Melanin
Deposition in Hair than for Maximal Growth
Peter J. B. Anderson, Quinton R. Rogers and James G. Morris
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA
ABSTRACT In 1986, the NRC recommended a dietary concentration of 4.0 g/kg of phenylalanine and 8.5 g/kg of
total aromatic amino acids for growing kittens on the basis of maximal growth rate and nitrogen balance. Black
hair-coated cats were given purified diets containing the following phenylalanine tyrosine (Phe Tyr) concen-
trations (g/kg diet): 4 2,44,46,48, 10 0, 10 2, 10 4, 10 6, 10 8, 10 10, 24 0 for at
least 6 mo. All other amino acids were present at about twice the requirements. Total melanin and the oxidation
product of eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA) were measured in hair. There was a significant linear
relationship between the concentrations of tyrosine in plasma and PTCA in hair. The relationship between PTCA
concentration in hair and Phe Tyr in the diet had a point of inflection at 16 g/kg Phe Tyr. Cats fed diets with
16 g Phe Tyr developed “red hair.” We confirmed the anecdotal reports that the black hair of cats can change
from black to reddish brown. An aromatic amino acid concentration 18 g/kg is recommended for the prevention
of visually discernible red hair in black-coated cats. Dietary concentrations 18 g total aromatic amino acids/kg
diet promote a greater ratio of PTCA:total melanin in hair. We are unaware of a secondary nutrient requirement
being so much greater than the requirement for growth. J. Nutr. 132: 2037–2042, 2002.
KEY WORDS: melanin eumelanin tyrosine phenylalanine cats
The color of mammalian hair results mainly from the se-
cretory products of the melanocytes. Organelles referred to as
melanosomes within these specialized dendritic cells synthe-
size melanin, which is secreted into the surrounding keratino-
cytes where they become incorporated into the hair. Follicular
melanocytes differ from those in the epidermis in that they
synthesize larger melanosomes, are active only during anagen
stages III–VI of hair growth, and are inactive during telegen
(1). Two types of melanin are synthesized in follicular mela-
nocytes: eumelanin, which is brown to black, and pheomela-
nin, which is reddish-brown (2). The pathways for the syn-
thesis of these two types of melanin were presented by Morris
et al. (3). The physiologic signals that regulate this switch
include the
-melanocyte–stimulating hormone and the ag-
outi protein (4). Dihydroxyphenylalanine (DOPA),
the hy-
droxylated product of tyrosine, is the precursor of both types of
melanin. We have been unable to find any reference to the
effect of dietary tyrosine (or phenylalanine) on the color of
mammalian hair. However, the tendency for phenylketonuric
human subjects to have fair hair has been commented upon
from time to time since the first observations of Folling in
1934 (5).
Rogers and Morris (6) demonstrated that phenylalanine
was an essential amino acid for growing kittens, but tyrosine
was dispensable when the diet contained adequate Phe. These
authors also demonstrated that the phenylalanine requirement
for maximal growth was not 7.5 g/kg diet in the presence of
10 g tyrosine/kg diet. Anderson et al. (7), using Latin-square
designs, showed that the phenylalanine requirement of grow-
ing kittens was not 5 g/kg diet in the presence of 5 g
tyrosine/kg diet. Further refinements to the aromatic amino
acid requirement of growing kittens were made by Williams et
al. (8) who reported a total requirement of 7.5 g/kg diet, of
which about half could be supplied by tyrosine. This latter
study used a Latin-square design of 10-d periods and based the
requirement on the minimal phenylalanine or phenylalanine
plus tyrosine for maximal growth and nitrogen balance. The
NRC (9) recommended a total aromatic amino acid require-
ment of 8.5 g/kg diet, with a minimum of 4.0 g phenylala-
nine/kg diet. The above values plus a slight overage for bio-
availability were used by the Association of American Feed
Control Officials (AAFCO) (10) for the Cat Food Nutrition
Profile used by the pet food industry. AAFCO (10) recom-
mended that diets for both kittens and adult cats should
contain 8.8 g phenylalanine plus tyrosine/kg diet with a min-
imum of 4 g phenylalanine.
Subsequent longer-term studies (11) demonstrated that di-
ets based on gelatin, containing amino acids in excess of the
NRC (9) recommendations, resulted in the coat hair of black
cats turning reddish-brown. The change in coat color was also
produced when cats consumed diets based on isolated amino
acids that included 12 g phenylalanine plus 4.5 g tyrosine/kg
Supported in part by Royal Canin, Centre de Recherche de Saint-Nolff,
Vannes, France.
To whom correspondence should be addressed.
Abbreviations used: AAFCO, Association of American Feed Control Offi-
cials; DAA, dispensable amino acid; DOPA, dihydroxyphenylalanine; EAA, essen-
tial amino acid; PH, phenylalanine hydroxylase; PTCA, pyrrole-2,3,5-tricarboxylic
acid; T
, triiodothyronine; T
, thyroxine; TSH, thyroid-stimulating hormone.
0022-3166/02 $3.00 © 2002 American Society for Nutritional Sciences.
Manuscript received 12 December 2001. Initial review completed 19 January 2002. Revision accepted 18 March 2002.
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diet. These levels were greatly in excess of those recommended
(9,10). However, when black cats consumed a diet that con-
tained 24 g phenylalanine/kg diet, and no tyrosine, there was
no change in hair color. These studies indicated that the
recommended (9,10) levels of phenylalanine and tyrosine
were inadequate to maintain black hair coat in cats.
The present study investigated the effects of a range of
dietary levels of phenylalanine and tyrosine on coat color in
black cats. Studies were conducted over periods of 69moto
assess the level of dietary phenylalanine and tyrosine required
to maintain black hair color.
Animals. Specic pathogenfree domestic short-hair cats and
kittens (n53) with black hair coats from the Feline Nutrition and
Pet Care Center, University of California at Davis were used. At all
times, cats and kittens consumed a puried diet and water ad libitum.
The experimental protocol was approved by the Animal Use and
Care Administrative Advisory Committee University of California,
Davis and conducted in accordance with the NIH guidelines (12) and
the Animal Welfare Act. Cats were housed either singly in 60 60
60 cm stainless steel metabolic cages or in pairs in 1.4 m
Room temperature was maintained at 21 2°C with a minimum
light:dark cycle of 14 h/10 h.
Diets. Eleven puried diets containing the following proportions
of phenylalanine and tyrosine, respectively (g/kg diet) were prepared:
42; 4 4; 4 6; 4 8; 10 0; 10 2; 10 4; 10 6; 10
8; 10 10; 24 0. Crystalline amino acids (Ajinomoto USA,
Teaneck, NJ) supplied the nitrogen in all diets, and the essential
amino acids with the exception of phenylalanine and tyrosine, were
supplied at 1.62 times the NRC (9) recommendations. The crude
protein (N 6.25) was held constant at 280 g/kg diet by using an
essential (EAA) and a dispensable (DAA) amino acid mixture. The
amount of the DAA mixture was adjusted for the nitrogen supplied
as phenylalanine and tyrosine. The EAA mixture contained (g/kg
diet): L-Arg HCl, 20; L-His HCl H
O, 6; L-Ile, 10; L-Leu, 24;
L-Lys HCl, 16; L-Met, 8; L-Cys, 7; L-Thr, 14; L-Trp, 3; L-Val, 12. The
DAA mixture contained (g/kg): L-Ala, 175; Gly, 175; L-Gln, 175;
L-Glu, 75; L-Asn, 150; L-Asp, 100; L-Pro, 150.
The constant ingredients in the diets were as follows (g/kg diet):
chicken fat (Foster Farms, Livingston, CA) 240; starch (Melojel,
National Food Starch and Chemical, Bridgewater, NJ) 275.5; sucrose,
100; cellulose, 20; mineral mixture 50 (8); vitamin mixture 5 (8);
choline chloride (Du Pont, Highland, IL), 4.5; sodium acetate (Fisher
Scientic, Santa Clara, CA) to balance the hydrochloride associated
with arginine, histidine and lysine; taurine (Taisho Pharmaceutical,
Torrance, CA) 2.5. The diets were blended in a 40-L food mixer,
which produced a diet with a consistency similar to cookie dough,
and were given to the cats in this form.
Experimental design
An insufcient number of black cats were available at one time to
supply all cats needed for the 11 dietary treatments. Therefore, cats
were allocated to one of four diet groups after they were accustomed
to a puried diet and were gaining body weight (Table 1). The ages
of the cats in group 1 ranged from 3 to 8 mo, those in groups 2 and
3, 23 mo and cats in group 4, 24 mo The groups are described
Group 1. Cats in this group had initial body weights ranging
from 2.1 to 4.1 kg; they were older than those in groups 24 and
included 13 females and 5 males. Treatments commenced on March
6, 1999 and continued for 269 d. The cats were allocated as follows
to the dietary treatments: Phe Tyr 4 2 (2 females); 4 4(2
females); 4 6 (2 females); 4 8 (1 male and 1 female); 10 0(2
females); 10 2 (4 males); 10 4 (2 females) and 10 6(2
Group 2. The 7 males and 4 females in this group had initial
body weights that ranged from 0.9 to 1.7 kg. Treatments commenced
on April 26, 1999 and continued for 227 d. The cats were allocated
to the dietary treatments as follows: Phe Tyr: 4 2 (1 male and
1 female); 4 4 (2 males); 4 6 (1 male); 4 8 (1 female); 10
0 (2 males); 10 2 (1 male and 1 female) and 10 6 (1 female).
Group 3. The 5 males and 5 females in this group had initial
body weights that ranged from 0.9 to 1.2 kg. Treatments commenced
on August 20 1999 and continued for 210 d. The cats were allocated
to the following dietary treatments Phe Tyr: 4 2 (four males); 4
6 (2 males); 4 8 (2 males); 10 2 (2 males) and 10 4(2
Group 4. The 5 females and 7 males in this group had initial
body weights of 0.8 to 1.5 kg and were allocated to the three dietary
treatments Phe Tyr: 10 8 (2 males and 2 females); 10 10 (3
males and 1 female) and 24 0 (2 males and 2 females). Treatments
commenced May 5, 2000 and continued for 191 d.
Food intakes were measured daily and body weights were recorded
weekly. Blood samples (3 mL) were drawn from the jugular veins of
unanesthetized cats into heparinized syringes every 12 mo. Plasma
was frozen at 80°C until analyzed for free amino acids. At the
beginning of each experiment, a rectangular area approximately 7
5 cm on the lateral abdomen of each cat was shaved. New hair
growth within this area was evaluated for the presence of red hairs
and then shaved at the time of each blood sampling and used for the
analysis of melanin or pyrrole-2,3,5-tricarboxylic acid (PTCA).
Analytical methods. Plasma amino acid concentrations were
determined on a Model 7300 Beckman Amino Acid Analyzer (0.4
cm 10 cm column packed with spherical cation exchange resin,
Beckman Instruments, Palo Alto, CA). Before analysis, plasma was
mixed with an equal volume of 0.28 mol/L sulfosalicylic acid. The
resulting precipitate was removed by centrifugation at 16,000 g.
Lithium hydroxide was added to an aliquot of the supernatant to
adjust the pH to 2.2 and the equivalent of 20
L of plasma was
injected onto the column of the analyzer.
Total melanin was determined according to the method of Ozeki
et al. (2) by dissolving 5 mg hair in 3 mL of Soluene-350 (Packard
Instrument, Meriden, CT). Once the hair was completely solubilized,
the optical density was determined in a Beckman Recording Quartz
Spectrophotometer (Beckman Instruments, Fullerton, CA) at wave-
lengths of 500 and 650 nm; the latter wavelength provides an index
of the proportion of pheomelanin in the hair. A standard curve for
total melanin was constructed using Sepia melanin standards (Sigma
Grouping of cats and number according to treatments
Treatment (phenylalanine
tyrosine g/kg diet) Group1Cats, n
10 012
10 214
10 412
10 612
10 844
10 10 4 4
24 044
1Refers to the group of cats assigned to the treatment.
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Chemical, St. Louis, MO) dissolved in Soluene-350. The CV of the
total melanin assay was 5.6% for black cat hair. Analyses were done
in triplicate.
The measurement of PTCA concentration in hair was based on a
modication of the peroxide oxidation method of Ito and Wakamatsu
(13). Briey, 10 mg hair was added to a mixture of 100
L deionized
water, 840
(1 mol/L) and 60
(30%) in a 20
125 mm screw-cap Pyrex culture tube (Corning Glass Works,
Corning, NY). The tubes and contents were placed in a 90°C water
bath until the hair was digested. After the sample had cooled to room
temperature, 20
(10%) and 500
L HCl (6 mol/L) were
added to the digest. The samples were twice extracted with 7 mL
diethyl ether, and extracts were pooled and evaporated under nitro-
gen. The residue was suspended in 200
L deionized water and 20
was injected onto a 4.6 mm 250 mm Xterra MS C
column (Waters, Milford, MA) heated to 55°C with a phosphate
buffered mobile phase at pH 2.1 containing 2% (v/v) methyl alcohol.
A Rainin LDC (Emeryville, CA)/Milton Roy Spectro Monitor 3000
detector (Rochester, NY) at a wavelength of 269 nm was used. The
retention time of the major peak area corresponded to that of the
PTCA (5) working standard, which was prepared according to the
method of Ito and Wakamatsu (13) and standardized by comparison
with a sample kindly supplied by Dr. Ito. The CV for the working
standard of PTCA was 1.1%, and for digested and ether-extracted
hair from a control cat, was 3.5%.
In addition to the objective measurements of melanin and PTCA,
the overall hair color of the cats was subjectively assessed by a visual
scoring system using a scale of 0 to 6 in which 0 black, 1 slight
brown tinge, 2 brownish black, 3 medium brown, 4 light
brown, 5 gray-light brown, 6 gray.
Statistical analyses. Data were analyzed by one-way ANOVA
and the post-hoc Tukey-Kramer multiple comparisons test. differ-
ences were considered signicant at P0.05.
The PTCA concentrations in hair of cats in dietary treat-
ments 4 2to102 in relation to the time they received
the diet are shown in Figure 1. The decline in concentration
of PTCA indicated that there was a decrease in eumelanin
concentration in hair. In contrast, in Figure 2, the mean
PTCA concentrations in the hair of the cats from the 10 6
to the 24 0 g/kg dietary treatments followed a positive trend
with time, indicating increasing concentrations of eumelanin
in hair. In this gure, the values for the cats in the 10 8 g/kg
group remained almost constant and the concentrations for
the 10 6 g/kg treatments were negatively related with the
time they received the diet. These observations indicate that
a concentration of 10 6 g/kg Phe Tyr is inadequate to
maintain black hair. This was supported by the visual assess-
ment of overall coat color (data not shown).
As would be anticipated, there was a general positive rela-
tionship between the concentration of Phe Tyr in the diet
and the concentration of tyrosine in plasma (Fig. 3). However,
it was not until the sum of Phe Tyr was 18 g/kg diet that
a marked elevation of the concentration of tyrosine in plasma
occurred. Values are also presented for redness score of the
new hair grown in the clipped area in relation to plasma
tyrosine concentration. The redness score for cats fed 18 g
total aromatic amino acids/kg diet was 0, indicating the ab-
sence of red hairs. Below this dietary concentration, cats had
increasing numbers of red hairs. The sum of the dietary con-
centration of Phe Tyr was also positively related to the
PTCA concentration in hair, but the relationship exhibited a
discontinuity at 16 g/kg. In Figure 4, the regression of
dietary concentration of Phe Tyr from 6 to16 g/kg diet on
PTCA concentration in hair had a lower slope, (and a lower
value), than for dietary concentrations of 1624 g/kg. The
point of intersection of the two linear phases occurred between
16 and 18 g Phe Tyr/kg diet.
When the values for PTCA concentration in hair for all
treatment groups were regressed on the concentrations of
tyrosine in plasma (Fig. 5), there was a signicant positive
relationship (r
0.87). This relationship indicates that
plasma concentration of tyrosine was the prime determinant of
the concentration of PTCA in hair and agrees with the rela-
tionship of the subjective visual assessment of red hairs in the
newly grown hair in the shaved patches and concentration of
plasma tyrosine presented in Figure 3.
The relationship between the concentrations of PTCA and
total melanin in hair for treatments of 1024 g Phe Tyr/kg
diet is presented (Fig. 6). At concentrations of 1016 g/kg
diet, the ratio was essentially similar, but at a concentration
FIGURE 1 Concentration of pyrrole-2,3,5-tricarboxylic acid
(PTCA) in the hair of cats fed puried diets containing the following
concentrations of Phe Tyr: 4 2, 4 4, 4 6, 4 8, 10 0, 10
2 and 10 4 (g/kg diet) at d 1, 85, 143, 252 and 281. Data from the
groups were combined. Upper and lower limits of the range are the
highest and lowest group mean PTCA concentrations on each day.
Values are means SEM. The numbers of cats in each group were: 4
28, 4 44, 4 65, 4 85, 10 06, 10 26, 10
FIGURE 2 Concentration of pyrrole-2,3,5-tricarboxylic acid
(PTCA) in the hair of cats fed puried diets containing Phe Tyr
concentrations of 10 6, 10 8, 10 10 and 24 0 (g/kg diet).
Results are means SEM,n4 for all treatments except 10 6, n
3.*Final concentrations differed (P0.05 and **P0.01) from that
of cats fed the 10 6 g/kg diet.
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18gofPheTyr/kg diet, there was an increase in the ratio
of PTCA to total melanin. This value coincided with the
dietary concentration at which there was a change in visual
assessment of hair color.
In addition to the loss of color, hair growth of cats fed
dietary Phe Tyr concentrations of 4 2and44 g/kg was
also compromised. The rst sign observed was alopecia sur-
rounding the mouth and eyes, which spread over the entire
facial area, then to the anks and limbs. Whiskers became
brittle and broke off giving the appearance that the cats had
been clipped. The hair on the body became rough, dry, gray
and thin, particularly on the anks and limbs. Because hypo-
thyroidism is associated with similar hair changes as those
observed in these cats, and as tyrosine is a precursor of the
thyroid hormones, a serum sample was taken from 4, 2, 7, 4,
and 3 cats receiving the Phe Tyr diets 4 2, 4 4, 4 6,
10 0, and 10 6, respectively, for measurement of thyroid
status. Free and total thyroxine (T
), free and total triiodo-
thyronine (T
), and thyroid stimulating hormone (TSH) were
measured by the Animal Health Diagnostic Laboratory, Mich-
igan State University, East Lansing. All of the values lay
within the normal range for cats. No signicant differences
between groups were found except for free T
. Free T
of cats receiving the Phe Tyr 4 6 g/kg diet were 3.7 0.4
pmol/l, (n 7) and differed (P0.01) from that of cats
receiving the Phe Tyr 10 0 diet [(1.8 0.3 pmol/L, (n
4)]. The reason for this difference is not known as these two
FIGURE 3 Relationships at the end of the study (6 mo) between
the sum of the dietary concentration of Phe Tyr and plasma concen-
tration of tyrosine and the coat color of the cats. A coat color score of
0black, 1 slight brown tinge, 2 brownish black, 3 medium
brown, 4 light brown, 5 gray-light brown, 6 gray. The coat score
of the cats fed the following diets differed (P0.05): 6 vs. 18, 20 and
24; 8 vs. 18, 20 and 24; 10 vs. 18, 20 and 24; 12 vs. 18, 20 and 24.
Plasma tyrosine concentrations in cats fed the following diets differed:
6, 8, 10 and 12 vs. 24 (P0.001); 14 vs. 24 (P0.05); 16 vs. 24 and
18 vs. 24 (P0.01). For the treatments (g Phe Tyr/kg diet) 6, 8, 10,
12, 14, 16, 18, 20 and 24, n8, 4, 11, 11, 6, 3, 4, 4, 4, respectively.
FIGURE 4 Relationship between the sum of the dietary concen-
tration of phenylalanine plus tyrosine and pyrrole-2,3,5-tricarboxylic
acid (PTCA) concentration in hair. Linear regressions were tted to the
two components of the plot, from 6 to 16 g and from 16 to 24 g total
aromatic amino acids/kg diet. The equations of the regressions and r
values are presented. For the treatments (g Phe Tyr/kg diet) 6, 8, 10,
12, 14, 16, 18, 20, and 24, n8, 4, 11, 11, 6, 3, 4, 4, 4, respectively.
FIGURE 5 Relationship between the concentrations of pyrrole-
2,3,5-tricarboxylic acid (PTCA) in the hair and tyrosine concentration in
the plasma of black hair cats given puried diets varying in phenylala-
nine and tyrosine concentrations. Values are means SEM,n3to8.
FIGURE 6 Relationship between the sum of the dietary phenyl-
alanine and tyrosine concentration and the concentrations of pyrrole-
2,3,5-tricarboxylic acid (PTCA) and total melanin in the hair of cats at
the end of the study. The ratio of PTCA:total melanin was less (P
0.05) for treatments 10 vs. 18, 20 and 24, and 12 vs. 18 and 24. For
the treatments (g Phe Tyr/kg diet) 10,12,14,16,18, 20, and 24, n11,
11, 6, 3, 4, 4, 4, respectively. Values are means SEM.
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groups received the same sum of phenylalanine tyrosine in
the diet. Hair growth was normal when the cats consumed
dietary Phe Tyr concentrations of 4 6 and 4 8 g/kg diet,
but the new growth was brown in color, not black, which was
reected in the PTCA concentrations that remained below 50
g/g hair. The greater Phe Tyr concentrations also resulted
in normalization of the whiskers. Neither feed intake (P
0.47) nor live weight gain (P0.39) was compromised in
cats given the Phe Tyr 4 2 and 4 4 g/kg diets compared
with cats given the other treatments.
Because all amino acids other than phenylalanine and
tyrosine were present at 1.62 times the level recommended
by the NRC (8), the low dietary Phe Tyr levels were
limiting hair growth. Plasma tyrosine levels were as low as 4.4
mol/L in cats given the Phe Tyr 4 2 g/kg diet.
This is very low compared with cats in our colony given a
commercial expanded diet (55 4.7
mol/L, n8). At this
latter level of plasma tyrosine, cat hair remains visually black.
After 6 mo, three of the four male cats in group 1
receiving the Phe Tyr 10 2 g/kg diet developed a
neurological condition involving the posterior limbs and tail.
On a slow walk, the cats had extended hind legs, resulting in
an uncoordinated gait. The tail was held vertically, bending
upward and forward over the back. On a fast walk, affected cats
hopped on their hind legs. The most severely affected cat was
hyperactive; it hypersalivated and emitted frequent vocaliza-
tions. Three affected cats were subjected to neurological ex-
amination. Conduction velocities of the peripheral nerves of
the hind limbs were measured using evoked potentials. Two
cats had 50% reductions in sensory nerve conduction veloc-
ity, but motor nerve conduction velocities were within the
normal range. Histologic examination of nerve biopsies
showed marked Wallerian degeneration of axons with second-
ary myelin collapse. A separate report will describe the neu-
rological ndings.
The most severely affected cat was transferred from the 10
2 g/kg diet to the 10 6 g/kg diet and within 1 mo, there
was a marked improvement in hind limb coordination, vocal-
ization was reduced and the hyperactivity disappeared. No
adverse clinical signs were observed in cats given diets with
phenylalanine tyrosine concentrations 10 6 g/kg.
This study demonstrated that black cats require higher
dietary Phe Tyr concentrations for the maintenance of coat
color than the values given by either NRC (9) or AAFCO
(10). Because black hair color is determined by the proportion
of the black-brown pigment eumelanin compared with the
reddish brown pigment pheomelanin, it appears that geneti-
cally determined eumelanin synthesis in cats requires a higher
concentration of Phe Tyr in the diet than the concentration
to maximize growth. Although both forms of melanin have a
common precursor (L-tyrosine), eumelanin is derived from
o-dihydroxyphenolic and indolic precursors alone, whereas
pheomelanin is formed by the conjugation of reactive phenolic
intermediates with L-cysteine, producing a benzothiazine in-
termediate. No studies on melanogenesis or its control have
been done in cats; however, in other species, Ito (14) reported
an inverse relationship between the two forms of melanin and
that the activity of tyrosinase controlled their relative propor-
tions. Low activities of tyrosinase led to pheomelanin biosyn-
thesis, whereas high tyrosinase activity led to eumelanin bio-
synthesis. A low activity of tyrosinase results in low levels of
dopaquinone, which is converted to glutathionyldopa and to
pheomelanin, whereas high activity of tyrosinase produces
excess dopaquinone, which inhibits glutathione reductase and
-glutamyl transpeptidase (enzymes required for pheomelano-
genesis). L-Tyrosine is not only a precursor of melanin but also
increases the activity of tyrosinase, thereby enhancing mela-
nin synthesis (15).
Using cultured melanocytes, it was shown (16) that the
pigmentation pattern changed with varying concentrations of
cysteine and tyrosine in the media. High tyrosine and low
cysteine concentrations favored eumelanogenesis. Because all
of the diets we used contained the same concentrations of Met
and Cys, the Cys pool in the plasma for the melanocyte should
have been adequate, and similar in cats across all treatments.
However, the concentration of tyrosine in plasma varied with
the dietary Phe Tyr concentration (Fig. 3) and presumably
the tyrosine pool available to the melanocytes reected these
changes. At low concentrations of dietary Phe Tyr, the
cysteine to tyrosine ratio in the pool would have favored
pheomelanin synthesis and a greater proportion of red hair.
With 16 g/kg of Phe Tyr, the concentration of tyrosine in
the plasma increased only slowly with increasing Phe Tyr,
whereas above 16 g Phe Tyr/kg diet, the rate of increase
in tyrosine in plasma was accelerated as illustrated in Figure 3.
Coincidental with this increase in plasma tyrosine, the inci-
dence of visually apparent red hairdisappeared (Fig. 3). In
addition, 16 g Phe Tyr/kg diet led to a marked increase in
the concentration of PTCA (an index of eumelanin) in hair
(Fig. 4). This conclusion is further supported by the signicant
positive relationship between the concentration of plasma
tyrosine and PTCA in hair (Fig. 5). Hair growth was also
compromised by low dietary concentrations of Phe Tyr.
Although black cat hair contains 184 2.4
mol Phe and 247
mol Tyr/g lipid-free dry matter (17), the loss from the
body through hair is trivial [2.5 mg Phe Tyr/(kg body
weight d)].
Tyrosine used for the synthesis of melanin may originate
from plasma tyrosine or from phenylalanine after hydroxyla-
tion with phenylalanine hydroxylase (PH). As a measure of
potentially available tyrosineto the follicular melanogenesis,
we used the sum of dietary phenylalanine and tyrosine. How-
ever, studies using nonfeline melanocyte cultures indicated
that phenylalanine may be a more efciently used source of
tyrosine for the follicular melanocyte than dietary tyrosine.
Epidermal melanocytes express mRNA for PH in association
with considerable enzyme activity. Cultured human epidermal
melanocytes in the presence of L-phenylalanine produce
40% more melanin than an equivalent concentration of
L-tyrosine (18). The transport of extracellular L-phenylalanine
and its intracellular metabolism to L-tyrosine by PH are cou-
pled with calcium transport, whereas L-tyrosine uptake by
melanocytes is calcium independent. The cofactor for PH,
6(R)-L-erythro 5,6,7,8, tetrahydrobiopterin, is produced de
novo and is recycled and regulated in melanocytes and kera-
tinocytes to control tyrosine hydroxylase, PH and tyrosinase
activity (19).
The considerably greater dietary intake of Phe Tyr re-
quired to maintain black hair in cats than for maximal growth
is consistent with the apparent K
values reported for reac-
tions involving tyrosine. The K
of the acyl synthetase is 4
mol/L (20), whereas the catabolic enzyme (tyrosine
aminotransferase) is 375 times greater (1.5 10
(21). Tyrosinase is the key enzyme that controls the biosyn-
thesis of melanin (22). A range of apparent K
values for
tyrosinase from murine melanomas has been recorded. In one
study, the apparent K
values for tyrosine and DOPA were 7
and 6 10
mol/L, respectively (23), and in a
subsequent study, apparent K
values of two isoenzymes of
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tyrosinase for tyrosine were 1.2 10
and 2.3 10
(24). K
values reported for tyrosinase isolated from cephalo-
pod ink were higher (1.7 10
and 10 10
respectively) for L-tyrosine and L-DOPA (25). The mamma-
lian K
value of tyrosinase for tyrosine indicates that the
concentration of tyrosine for melanin synthesis would have to
be 3 to 17 times higher than that required for growth.
Our study demonstrating that melanin in cat hair was
positively related to the concentration of dietary tyrosine with
a constant (10g/kg) phenylalanine concentration, does not
support the conclusions of Schallreuter (19) who asserted that
the active transport of L-phenylalanine and its autocrine
turnover to L-tyrosine via PH in the cytosol of the epidermal
human melanocyte provides the majority of the L-tyrosine pool
for melanogenesis.
The observation that the clinical signs of hyperactivity and
ataxia resolved or were reduced after the most severely affected
cats were given the 10 6 g/kg diet suggests the involvement
of tyrosine in catecholamine production rather than structural
nerve degeneration. In catecholamine synthesis, the conver-
sion of tyrosine to DOPA is catalyzed by tyrosine hydroxylase,
which is the rate-limiting enzyme of the pathway. Isolates of
the enzyme from the preoptic region and hypothalamus of rats
both gave apparent K
values of 8 10
mol/L for tyrosine
(26,27), which is considerably lower than the K
for tyrosi-
nase, but about twice the K
of the acyl synthetase. Other
factors in addition to the K
value, such as transport, may
have a role in determining catecholamine synthesis. Tyrosine
deciency is manifested in multiorgan systems, including a
reduction in hair growth and follicle density, hair color and
peripheral sensory neuropathy, which is probably related to
the origin in the vertebrate embryo of the skin melanocytes
and hair bulbs in the neural crest (28).
Red coat has been described in dogs and cats given certain
commercial and therapeutic foods. However, reports of its
occurrence have been mainly anecdotal, such that it has
generally been considered to be a myth or an old wives tale
(29). The present study indicates that eumelanin production
in cats is compromised if the sum of readily available dietary
Phe Tyr is 16 g/kg diet. We recommend that the dietary
phenylalanine plus tyrosine requirement be increased to at
least 18 g of available aromatic amino acids/kg diet to
maintain black hair color in cats. As a diagnostic aid, a plasma
tyrosine concentration of 50
mol/L is necessary to prevent
red hairin cats.
We thank Shosuke Ito, Fujita Health University School of Health
Sciences, Toyoake, Japan for providing the PTCA standard we used,
Debbie Bee for expert care of the cats and Roche Products Nutley, NJ
for the gift of the vitamin mixture.
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... A number of publications have highlighted the nutritional connection, with much of the work focused on feline and canine models. For example, in cats, a telling discovery was made that the intake of the aromatic amino acids Tyr and phenylalanine (Phe) required for normal, healthy growth and development was insufficient to support maximal expression of melanins in the hair Anderson et al., 2002). These studies have illustrated the importance of adequate Phe and Tyr intake for the biosynthetic processes responsible for melanin synthesis. ...
... Although the primary influence on hair colouration in animals is genetic (Robinson, 1991), a number of exogenous factors are also known to exert an effect (Busch-Kschiewan et al., 2004). The evidence for a nutritional impact has been accumulating over recent times; with intake of the amino acids tyrosine, phenylalanine and cysteine, as well as copper all having been implicated in studies (Anderson et al., 2002;Watson et al., 2015Watson et al., , 2017. The black coat of certain animals is known to discolour, often showing patches of brown or reddening (Yu et al., 2001). ...
... Apart from being an aesthetic concern for breeders and owners, research into the phenomenon has suggested that it may have implications for differentiating between adequate versus optimal nutritional requirements. For example Anderson et al. (2002) for kittens and Watson et al. (2015) for puppies both highlighted that nutritional requirements for growth appear insufficient to also meet the demands of regular melanin synthesis. ...
Full-text available
Although the principle determinant of melanin derived hair colour and patterning in mammals is genetic, environmental factors are now also thought to play a role by influencing the expression of the inherited component. It has been demonstrated, for example, that the concentration of melanins deposited in the hair of cats is influenced by the amino acid composition of their diets. The observation has since been extended to dogs, whereby puppies were found to require Tyrosine intake significantly greater than that recommended for normal growth and development in order optimise melanin expression in their coat. Much of the work to date has been conducted in growing animals. These animals might, as a consequence, be considered to operate under additional nutritional strain. Less is known about the relationship between nutrition and hair melanin deposition in healthy adult animals. In this study we demonstrate for the first time that colour expression in the hair-coat of dogs is dependent on dietary intake of Tyrosine, and that the requirement appears to be in excess of the minimum level recommended to maintain health. Using spectrophotometry we were able to show that dogs fed 5.6g/Mcal Phe/Tyr showed reduced dilution of their black coat pigment compared to dogs fed 3.5g/Mcal Phe/Tyr. Specifically, following 16 weeks at the higher Tyr intake, dogs showed less yellow pigmentation to their coat (P=0.0032), and after 24 weeks at the higher intake dogs showed less red (P<0.0001) and yellow (P<0.0001), as well as greater overall dark pigmentation (P<0.0001).
... It has also been shown that another pathway of L-Tyrosine and, therefore, Phenylalanine metabolism leads to the production of Melanin, a fundamental pigment for maintaining the color of the hair. In fact, appropriate intake of this EAA contributes, both in dogs and in cats, to the development and maintenance of optimal hair color [73][74][75]. ...
Full-text available
Dry pet food, made of fresh meats and especially meat meals, represents one of the main types of complete food available on the market by virtue of its practicality and long shelf life. The kibble production process includes mixed thermal and mechanical treatments that help to improve the palatability and durability of the final product but may have undesirable effects on nutrient bioavailability and digestibility. An analysis of the protein and lipid content of different dry pet food formulations, together with an in vitro digestibility analysis, can reveal which formulation can provide a more nourishing diet for pets. In this study, a quantitative and qualitative analysis was performed on three different formulations of chicken-based dry pet food, consisting of fresh meats, meat meals, or a mix of these two. The soluble protein concentration was determined by the Bradford assay, while the crude protein content was assessed through the Kjeldahl method. Quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS) was used to analyze the amino acid (AA) and lipid compositions. Finally, a gastric and small intestinal digestion simulation was used to determine the in vitro digestibility. The results show that dry pet food consisting only of chicken fresh meats has the highest content of soluble protein; it also contains more Essential AAs, Branched-Chain AAs, and Taurine, as well as a greater quantity of monounsaturated and polyunsaturated fatty acids. In addition, its in vitro digestibility was the highest, exceeding 90% of its dry weight, in agreement with the soluble protein content. These findings thus make the fresh-meat-based formulation a preferable choice as dry pet food.
... The deficiencies of some micronutrients can also affect the coloration of the hair (Morris, 2002). The recent experiments have shown that a diet with high-dose tyrosine and phenylalanine increases the intensity of black hair in predators (Anderson et al., 2002;Watson et al., 2015Watson et al., , 2017Watson et al., , 2018. The distribution of color variants of foxes on different Kuril Islands is not the same (Ishino, 1925;Klumov, 1960;Voronov, 1974). ...
... Hormonal regulation of melanin-based coloration and any correlated traits are of interest to behavioral endocrinologists because melanin is often a key component of social signals (Majerus, 1998;Jawor and Breitwisch, 2003;Roulin, 2004;Ducrest et al., 2008;McGraw, 2008). Expression of melanin pigments, which are synthesized from nutritionally dispensable amino acids (e.g., tyrosine; Anderson et al., 2002), can be affected by physiological, genetic, and social mechanisms (Thody 1999;Jawor and Breitwisch, 2003;Roulin, 2016). ...
Hormones can mediate suites of correlated traits. Melanocortins regulate melanin synthesis and elements of the melanocortin system can directly, and indirectly, affect a number of other traits, such as stress reactivity. Trait correlation in with the melanocortin system have been studied mainly in birds and mammals but less so in reptiles. We examined adult male western fence lizards (Sceloporus occidentalis) and if melanization was correlated with plasma levels of three hormones, including peptide hormone α-melanocyte stimulating hormone (α-MSH), testosterone and corticosterone, and ectoparasite loads. This lizard is darker at higher elevations in California, and we compared five high-elevation and four low-elevation populations during comparable periods of the breeding season at each site. We first validated use of an α-MSH assay kit with lizard plasma. Since Anolis carolinensis is one of the few species with published values for α-MSH plasma levels, we assayed both Anolis and Sceloporus plasma and compared hormone values to those we generated for Anolis to the publish values. We also evaluated effects of different methods of storing spiked plasma pools on resulting α-MSH concentrations. Plasma levels of α-MSH did not differ significantly, but some populations differed significantly in mean corticosterone and mean testosterone. Combining all individuals from the nine populations, we found that individual variation in α-MSH was not associated with individual variation in melanization, but levels of α-MSH were positively associated with plasma testosterone and negatively associated with corticosterone. The lack of association between individual levels of melanization and expression of most other traits differs from a growing number of within-population studies of melanization, and we discuss what differences in physiological mechanisms could produce different hypothetical patterns. Circulating levels of -MSH are only one element of the melanocortin system; in situ synthesis of α-MSH by the skin and the diversity of melanocortin receptors could also contribute to variation in traits mediated by the melanocortin system and should be examined.
... Synthetic pathways toward phaeoand eumelanogenesis are thought to differ, most notably in the role of the amino acid cysteine, which acts in phaeomelanin synthesis only (Land and Riley 2000). Amino-acid availability in the diet affects melanin distribution and coat color in cats (Anderson et al. 2002), but no comparable study is yet available in birds. We do know that factors like circulating sexhormone levels in Mallards affect feather-melanin composition, where phaeomelanin content in certain body regions is elevated in birds having higher testosterone titers (Haase et al. 1995). ...
The carotenoid-pigmented bill of Zebra Finches (Taeniopygia guttata) has received much recent attention as a sexually selected signal of quality, but these birds also display several sexually dichromatic plumage traits, including rust-colored cheek patches, a black breast band, and brown flanks. Black, brown, and earth-toned features in animals are thought to be produced by melanin pigments, but few studies have identified the melanin content of such colors in bird feathers. We used a series of biochemical techniques to investigate the pigmentary basis of these plumage colors in male Zebra Finches. All three feather traits contained melanin pigments, but varied in the amounts of the two basic forms of melanin (eumelanin and phaeomelanin). Black breast feathers contained predominantly eumelanin, whereas cheek and flank feathers contained extraordinarily high concentrations of phaeomelanin. Conventional methods of carotenoid analysis detected no carotenoids in either the cheek or flank feathers. Coloración Basada en Melaninas en las Plumas Ornamentales de los Machos de Taeniopygia guttata Resumen. El pico pigmentado con carotenoides de Taeniopygia guttata ha sido destacado recientemente como una señal de calidad seleccionada sexualmente, pero estas aves también presentan varios caracteres de plumaje sexualmente dicromáticos, incluyendo parches en las mejillas de color óxido, una faja pectoral negra y flancos de color café. Se cree que las tonalidades negras, cafés y color tierra son producidas por melaninas en los animales, pero existen pocos estudios que hayan identificado el contenido de melanina de dichos colores en las plumas de las aves. En este estudio empleamos una serie de técnicas bioquímicas para investigar la base pigmentaria de estos colores del plumaje en machos de T. guttata. Los tres caracteres de las plumas contaron con pigmentos melánicos, pero variaron en las cantidades de las dos formas básicas de melanina (eumelanina y feomelanina). Las plumas negras del pecho presentaron principalmente eumelanina, mientras que las de las mejillas y los flancos presentaron concentraciones extraordinariamente altas de feomelanina. Los métodos tradicionales de análisis de carotenoides no detectaron este tipo de pigmentos en las plumas de las mejillas y los flancos.
Domestic cats (carnivores) require high amounts of dietary amino acids (AAs) for normal growth, development, and reproduction. Amino acids had been traditionally categorised as nutritionally essential (EAAs) or nonessential (NEAAs), depending on whether they are synthesized de novo in the body. This review will focus on AA nutrition and metabolism in cats. Like other mammals, cats do not synthesize the carbon skeletons of twelve proteinogenic AAs: Arg, Cys, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Like other feline carnivores but unlike many mammals, cats do not synthesize citrulline and have a very limited ability to produce taurine from Cys. Except for Leu and Lys that are strictly ketogenic AAs, most EAAs are both glucogenic and ketogenic AAs. All the EAAs (including taurine) must be provided in diets for cats. These animals are sensitive to dietary deficiencies of Arg and taurine, which rapidly result in life-threatening hyperammonemia and retinal damage, respectively. Although the National Research Council (NCR, Nutrient requirements of dogs and cats. National Academies Press, Washington, DC, 2006) does not recommend dietary requirements of cats for NEAAs, much attention should be directed to this critical issue of nutrition. Cats can synthesize de novo eight proteinogenic AAs: Ala, Asn, Asp, Gln, Glu, Gly, Pro, and Ser, as well as some nonproteinogenic AAs, such as γ-aminobutyrate, ornithine, and β-alanine with important physiological functions. Some of these AAs (e.g., Gln, Glu, Pro, and Gly) are crucial for intestinal integrity and health. Except for Gln, AAs in the arterial blood of cats may not be available to the mucosa of the small intestine. Plant-source foodstuffs lack taurine and generally contain inadequate Met and Cys and, therefore, should not be fed to cats in any age group. Besides meat, animal-source foodstuffs (including ruminant meat & bone meal, poultry by-product meal, porcine mucosal protein, and chicken visceral digest) are good sources of proteinogenic AAs and taurine for cats. Meeting dietary requirements for both EAAs and NEAAs in proper amounts and balances is crucial for improving the health, wellbeing, longevity, and reproduction of cats.
The dog has assumed a prominent role in human society. Associated with that status, diet choices for companion dogs have begun to reflect the personal preferences of the owners, with greater emphasis on specialty diets such as organic, vegan/vegetarian, and omission or inclusion of specific ingredients. Despite consumer preferences and many marketing strategies employed, the diets must ensure nutritional adequacy for the dog; if not, health becomes compromised, sometimes severely. The most frequent consideration of consumers and dog food manufacturers is protein source and concentration with a growing emphasis on amino acid composition and bioavailability. Amino acids in general play diverse and critical roles in the dog, with specific amino acids being essential. This review covers what is known regarding amino acids in dog nutrition.
Coat color is an obvious phenotypic characteristic. When coat color changes, it is readily observable and may be a cause for concern among veterinary clients. Some coat color changes are expected. Consider, for example, those feline breeds with color‐point coat patterns. Siamese and Tonkinese cats are born white and develop highlights of color at extremities as they age. Those unfamiliar with the breed and/or new breeders may benefit from education concerning normal development so that they have appropriate expectations. Other coat color changes, while expected, are an adverse response to medical therapy. For instance, a late effect of radiation therapy in many companion animal patients is leukotrichia, the whitening of fur in irradiated regions of the body. Other coat color changes are equally drastic, such as the stark transformation of black‐coated dogs and cats into red or rust‐brown. Some of these changes are reversible. Others, such as age‐related graying of the muzzle, are not. Most changes are strictly cosmetic; however, salivary and tear staining indicate underlying conditions that may require medical management. One cannot underestimate the importance of taking a thorough dietary history, as well as performing a comprehensive physical examination. In situations where underlying disease exists, these tools may provide clues as to the nature of the problem and what, if anything, the clinician can do to manage, if not resolve, the issue.
We have previously determined Phenylalanine (Phe) requirements in mature dogs; however, little information is available on differences of Phe minimum requirements (MR) on different breed sizes. The objective of this study was to determine Phe requirements in adult dogs of 3 different breed sizes using the direct amino acid oxidation (DAAO) technique. In total, 12 adult dogs were used, 4 Miniature Dachshunds (5.3 ± 0.6 Kg BW; 1.8 ± 0.1 years old; mean ± SD), 4 Beagles (8.3 ± 0.7 Kg BW; 6.7 ± 0.2 years old; mean ± SD), and 4 Labrador Retrievers (34.9 ± 2.2 Kg BW; 4.4 ± 1.4 years old; mean ± SD). A basal Phe-deficient diet with excess of Tyrosine (Tyr) was formulated. Dogs were randomly fed the basal diet supplemented with increasing levels of Phe; the Phe content in the final experimental diets was 0.24, 0.29, 0.34, 0.44, 0.54, 0.64, and 0.74%. After 2 d of adaptation to the experimental diets, dogs underwent individual DAAO studies. During the DAAO studies, total daily feed was divided in 13 equal meals; at the sixth meal, dogs were fed a bolus of L-[1-¹³C]-Phe (9.40 mg/kg BW), and thereafter, L-[1-¹³C]-Phe (2.4 mg/kg BW) was supplied with every meal. Total production of ¹³CO2 (¹³CO2) during isotopic steady state was determined by enrichment of ¹³CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The mean requirement for Phe and the 95% confidence interval (CI) were determined using a 2-phase linear regression model. To account for differences in feed intake, requirements were expressed in BW⁻¹.d⁻¹. The mean requirement for Phe were 41.9, 41.3, and 42.6, and upper 95% CI of Phe requirements were 57.3, 58.4, and 64.8 BW⁻¹.d⁻¹ for Miniature Dachshunds, Beagles, and Labrador Retrievers, respectively. The mean requirement and the upper 95% CI for the pooled data (all dogs) was 45.3 and 55.4 BW⁻¹.d⁻¹, respectively. In conclusion, the Phe requirements for different breeds were similar among dog breeds studied. However, Phe recommendations proposed in this study are lower than those proposed by NRC and AAFCO ( BW⁻¹.d⁻¹).
Full-text available
We investigated the effect of varying concentration of 1-tyrosine and 1-cysteine in culture medium on melanin production by human skin melanocytes (skin phototype II/III). In addition to the analyses of dopa oxidase activity and total melanin, pheomelanin production in the cells was assessed by high-performance liquid chromatography determinations of pheomelanin degradation products, 3-aminotyrosine and 4-amino-3-hydroxyphenylalanine. As another marker for pheomelanin, melanosomal sulfur was determined by the use of X-ray microanalysis. With varying concentration of both amino acids, profound changes in the pigmentation patterns of the melanocytes were observed. A high concentration of 1-tyrosine (0.2 mM) was always connected with increased pigmentation. In combination with a low 1-cysteine content we saw an increase in tyrosinase activity and the highest melanin content. At high concentrations of both 1--tyrosine and 1-cysteine, the melanocytes showed reduced tyrosinase activity and they produced notably more pheomelanin. In case of the pheomelanin measurements by high-performance liquid chromatography and the sulfur detection with X-ray microanalysis, strongly increased concentrations were found when cells were maintained in high 1-tyrosine medium as compared with those grown with low 1-tyrosine. This was especially true for the combination with low 1-cysteine showing that the 1-tyrosine content of the medium strongly influences not only the eumelanin but also the pheomelanin production in the cultured melanocyte. It can be concluded that variations in the concentrations of 1-tyrosine and 1-cysteine in culture medium can be used to regulate the melanogenetic phenotype under in vitro conditions.Keywords: pheomelanin, X-ray microanalysis
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The effect of deleting each of the amino acids known to be essential for the young rat was determined in post weanling kittens fed a purified diet containing only L-amino acids as the source of dietary nitrogen. When any one of the 10 amino acids (arginine, lysine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, valine) were deleted from the diet food intake decreased, the kittens lost weight, and there was a dramatic drop in each corresponding amino acid in the blood plasma; indicating that each of the above amino acids is essential for the kitten. Deletion of all the amino acids except the 10 essential amino acids plus alanine resulted in a decreased weight gain to about 1/3 normal; indicating that although all the other amino acids could be synthesized, one or more of the dispensable amino acids may be required for maximal growth. When any one of the essential amino acids was decreased to one-half that present in the basal diet, there was no decrease in weight gain, indicating that the high protein requirement of the kitten is not the result of an unusually high requirement for the essential amino acids.
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The role of catecholaminergic mechanisms in determining the changes in the rat's preoptic cyclic adenosine monophosphate (cAMP) concentration during sleep deprivation and recovery induced by ambient temperature was investigated in the present study. To this end, the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, was measured in the preoptic region of rats maintained in: (a) control (22 degrees C for 52 h), (b) deprivation (-10 degrees C for 52 h), and (c) recovery (22 degrees C for 4 h after 48 h at -10 degrees C) conditions. The enzyme followed a Michaelis-Menten kinetic. The analysis of substrate-related kinetic parameters (Km and Vmax) did not show any clear-cut difference between experimental conditions, which, as already known, induce both sleep deprivation and recovery in relation to significant cAMP changes.
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The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.
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Two experiments were conducted to determine the phenylalanine requirement of the kitten and the ability of tyrosine to substitute for phenylalanine in the diet. In both studies purified L-amino acid diets were presented to six male and six female specific-pathogen--free kittens for six experimental periods of 10 d each, according to a 6 X 6 latin square design. In the first experiment, kittens were given tyrosine-free diets with either 4.0, 6.5, 7.5, 8.5, 9.5 or 15.0 g phenylalanine/kg diet. Mean daily weight gain and N retention were maximal at 7.5 g phenylalanine/kg diet. In the second experiment, diets contained 10.0 g tyrosine/kg diet, plus 1.5, 2.5, 3.5, 4.5, 5.5 or 10.0 g phenylalanine/kg diet. Mean daily weight gain and N retention were maximal at 3.5 g phenylalanine/kg diet. This indicates that the dietary phenylalanine requirement of the kitten is 7.5 g phenylalanine/kg diet, and that about half of this requirement may be replaced by tyrosine.
The articles published by the Annals of Eugenics (1925–1954) have been made available online as an historical archive intended for scholarly use. The work of eugenicists was often pervaded by prejudice against racial, ethnic and disabled groups. The online publication of this material for scholarly research purposes is not an endorsement of those views nor a promotion of eugenics in any way.
Melanocytes produce two chemically distinct types of melanin pigments, eumelanins and pheomelanins. These pigments can be quantitatively analyzed by acidic KMnO4 oxidation or reductive hydrolysis with hydriodic acid (HI) to form pyrrole-2,3,5-tricarboxylic acid (PTCA) or aminohydroxyphenylalanine (AHP), respectively. Dark brown melanin-like pigments are also widespread in nature, for example, in the substantia nigra of humans and primates (neuromelanin), in butterfly wings and in the fungus Cryptococcus neoformans. To characterize such diverse types of melanins, we have improved the alkaline H2O2 oxidation method of Napolitano et al. (Tetrahedron, 51: 5913–5920, 1995) and re-examined the HI hydrolysis method of Wakamatsu et al. (Neurosci. Lett., 131: 57–60, 1991). The results obtained with H2O2 oxidation show that 1) pyrrole-2,3-dicarboxylic acid (PDCA), a specific marker of 5,6-dihydroxyindole units in melanins, is produced in yields ten times higher than by acidic KMnO4 oxidation, and 2) PTCA is artificially produced from pheomelanins. The results with HI hydrolysis show that dopamine-melanin produces a 1:1 mixture of 3-amino and 4-amino isomers of aminohydroxyphenylethylamine, while the isomer ratio is about 0.2 in melanins prepared from dopamine and cysteine. These results indicate that alkaline H2O2 oxidation is useful in characterizing synthetic and natural eumelanins and that reductive hydrolysis with HI can be applied to analyzing oxidation products of dopamine such as neuromelanin.
A highly sensitive and reliable assay for tyrosine hydroxylase (TH) activity in hypothalamic homogenates of male rats using high-performance liquid chromatography with electrochemical detection is described. Modification of sample preparation and chromatographic conditions led to a complete separation of L-3,4-dihydroxyphenylalanine (L-DOPA) and alpha-methyldopa from all interfering catecholamines and their metabolites. This assay is highly sensitive; 2 pmol of L-DOPA formed enzymatically could be measured. We were able to determine TH activity in tissue pieces weighing less than 1 mg. TH activity was not changed after storage for three months at -80 degrees C. In hypothalamic homogenates the Michaelis constant (KM) for L-tyrosine was 80.5 +/- 6.5 mumol/l and the maximal velocity (Vmax) was 132.5 +/- 10.5 pmol/mg of protein per min for L-DOPA formed enzymatically.
We describe results demonstrating the positive regulation of melanogenesis by two substrates of the melanogenic pathway. We have found that L-tyrosine and L-dihydroxyphenylalanine (L-dopa), whose metabolic fates are affected by the activity of that pathway, can also act as its regulators. In living pigment cells, tyrosinase (EC, a crucial and rate-limiting enzyme of melanogenesis, acts in subcellular organelles known as melanosomes. Melanin is laid down only in these organelles. We demonstrate that supplementing Ham's F-10 medium with additional L-tyrosine or L-dopa during the culture of amelanotic Bomirski hamster melanoma cells results in a rapid increase in melanin formation, which is not simply due to greater availability of substrate. There is a rapid increase in tyrosinase activity and a large scale synthesis of melanosomes. The effects of L-tyrosine and L-dopa are prevented by the addition of cycloheximide. The actions of L-tyrosine and L-dopa are specific in that under similar conditions D-tyrosine, D-dopa, N-acetyl-L-tyrosine, L-phenylalanine, L-tryptophan and L-valine have little or no effect. The two substrates, L-tyrosine and L-dopa, appear to act through related but distinct mechanisms. Our findings provide an example of a little-known phenomenon: regulation of a differentiated eukaryotic phenotype through positive control by substrates in the pathway.