subjected to a much more extensive Zn**
analysis. The Zn®> concentrations found
in the various organs of this animal are
listed in Table 2. It may be noted that
the Zn®* concentration in the flesh, fat,
and bone samples are about twice those
observed in the samples of the previous
year (see Table 1). It is also apparent
that hair, liver, and bone concentrate
zinc to a greater extent than the other
organs.
The observation of these Zn®5 concentrations in farm-produce samples sug-
gested the possibility that Zn®> could be
measured in individuals obtaining their
food supply from these irrigation proj-
ects. For the measurement of Zn® in
people, a 3- by 5-in. sodium iodide crys-
tal detector operated in a shielded room
similar to the installation developed at
Argonne National Laboratory (2) was
used. The gamma-ray spectrum ofan in-
dividual who consumed approximately
0.1 and 0.7 kg, respectively, of the meat
burden for this isotope (4). Also, even
the highest Zn%5 value in Table 1 (for
pasture grass) is less than 5 percent of
the maximum permissible concentration
for human foods (4).
Smaller amounts of the radioisotopes
Cr5? and Sc*6 have been detected in samples of pasture grass but have not been
found in farm produce.
R. W. Perkins
J. M. Nre_sen
Hanford Laboratories O peration,
General Electric Company,
Richland, Washington
References
1. J. J. Davis and R. W. Perkins, unpublished.
2. C. E. Miller ef af., Nucleonics 14, No. 4, 40
(1956).
3.
. Argonne National Laboratory Rept.
No. ANL-57535 {July (957), pp. 53-56.
4. “Recommendation of the International Commission on Radiological Protection,’ @rit. f.
Radiol, Suppl. No. 6 (1955).
23 September 1958
and milk per day from the sourceslisted
in Table 1 is shown in Fig. 1. This Zn®
phoiopeak area represents about 3.6.x
10-2 we or 80,000 disintegration/min.
Studies on 4-Keto-L-Proline
ured whose diet did not include food
from the irrigation project but did in
some cases include drinking water whose
source was the Columbia River. The
gamma-ray spectra of most of the individuals whose drinking water originated
Abstract. The administration of ketoproline to chick embryos resulted in an
increase in the free hydroxyproline. This
phenomenonis explained by the inhibitory
action of ketoproline on the catabolism of
Twelve other individuals were meas-
in the Columbia River show a small Zn®5
photopeak. The Zn®5 content of these individuals was estimated to be between
5000 and 10,000 disintegration/min. The
gamma spectra of individuals who re-
ceive neither their water nor their food
supply from the Columbia Riverorits
irrigation projects showed no detectable
Zn*> photopeaks. The Zn®foundin all
samples of foods, in fodder, or in individuals could be traced either to Colum-
sion of the former to the latter. Ketoproline was found to be reduced to hydroxyproline by the supernatant fraction of
rat-kidney homogenate in the presence of
a reducedpyridine nucleotide.
Since hydroxyproline occurs uniquely
in collagen in the animal, studies on the
biogenesis and metabolism of this compound have a great significance in the
understanding of the biochemistry of col-
lagenous tissues. In the course of a sur-
vey of compoundsstructurally related to
hydroxyproline for their ability to affect
counting device of the Argonne National
administration of 4-keto-1-proline (7) to
chick embryos was found to produce an
Laboratory type (2) Zn®> could be measured in most individuals who receive
their drinking water from the Columbia
River downstream from Hanford, provided that some purification step in the
local water treatment does not remove
the isotope.
Zinc-65 has been reported (3) in indi-
viduals living near the Pacific Proving
Grounds and was shown to be a result
of contamination from nuclear tests;
however, Zn® from nuclear tests has not
been observed in foods raised in this
country. The presence of Zn*®in the
amounts observed here in no way con-
stitutes a hazard. Even the value of
3.6 x 10°? wc of Zn®> in the individual
whose diet included the meat, milk, and
drinking water of Table 1, is less than
0.01 percent of the total permissible body
9 JANUARY 1949
was found to be twofold: (i) inhibition
of hydroxyproline destruction and {ii) ©
conversion of ketoproline to hydroxyproline.
A strain of Achromobacter grown on
hydroxyproline as the sole source of
carbon metabolized hydroxyproline extensively. However, when increasing
amounts of ketoproline were added to
the incubation mixture, destruction of
hydroxyproline was diminished (Table
2). Proline was also found to antagonize
the metabolism of hydroxyproline under
similar conditions. However, the me-
tabolism of proline by proline-adapted
Achromobacter was not appreciably af-
fected by ketoproline, Similar observations were made when mammalian liver
or kidney mitochondria were used in
place of the bacteria. Neither the bacteria nor the mitochondria were able to
convert ketoproline to hydroxyproline in
any detectable amounts.
When ketoproline was incubated with
a well-dialyzed soluble fraction of rat-
kidney homogenate, it was reduced to
hydroxyproline as well as by the conver-
bia River drinking water or to food from
the lower irrigation projects. It is believed that, with a sensitive total-body
a period of several hours. Although administration of 5 mg of hydroxyproline
produced a comparable increase, this
lasted for less than 2 hours. The mechanism by which ketoproline produces the
increase in free hydroxyproline in vivo
the metabolism of this imino acid, the
Table 1. Effect of ketoproline on free proline and hydroxyproline levels in chick
embryos. Twelve-day-old embryos received either 0.2 ml of saline or 5 mg of
ketoproline in 0.2 ml of water. After 24
hours, 80-percent ethanol extracts of the
embryos were assayed for hydroxyproline
(3) and proline (6).
Treatment
Control
(av. of 6)
Ketoproline
administered
(av. of 4)
Wer
Free
wt.of hydroxyembryo proline
Free
proline
Free
hydroxyproline /
proline
{g)
(ug)
(ug)
free
4.2
112.1
206.2
1.84
6.3
627.2
218.7
0.35
increase in free hydroxyproline.
For these studies 5 mg of free 4-keto-
L-proline in 0.2 ml of water was placed
in the air space of a 12-day-old chick
embryo through an opening in the shell.
After 24 hours an 80-percent ethanol ex-
tract was prepared from the whole em-
bryo and hydroxyproline was assayed
colorimetrically by a modification (2)
of the method of Neuman and Logan
(3), The results are shown in Table 1.
Table 2. Effect of increasing concentrations
of ketoproline
on hydroxyproline
metabolism by Achromobacter. One milligram of hydroxyproline was incubated for
30 minutes with 9.4 mg (dry weight) of
hydroxyproline-adapted
Achromobacter
(7).
Ketoproline/
Hydroxyproline
Inhibition
(wg)
“
It can be seen that ketoproline caused a
hydroxy-
remaining
proline without affecting the proline
level. Similarly, when [5 mg of ketoproline was administered subcutaneously to
rats (200 to 250 ¢), a prolonged elevation of the blood hydroxyproline level
was observed, an increase from 0.55 to
1.0 mg per 100 ml being maintained for
0
]
2
3
4
3
97
340
420
585
690
780
five- to sixfold increase in free hydroxy-
proline
(Go)
29
36
54
66
76
95