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Hypercholesterolemia in experimental and human hepatic porphyria
Hypercholesterolemia in Experimental and Human Hepatic Porphyria BY LUIGI
TADDEINI, KAREN L. NORDSTROMAND
bility also exists that AIA and DDC enhances the production of one or more enzymes of cholesterol synthesis, in a way analogous to what Granick and Urata have demonstrated to occur for the enzyme S-aminolevulinic acid synthetase of the porphyrin synthetic pathway. The chemicals, in fact, may have their primary effect on the genetic control of the rate of synthesis of different proteins by the liver. A review of the records of 51 patients with hepatic porphyria, in whom serum cholesterol determinations were recorded, reveals that hypercholesterolemia, although inconstant, is of common occurrence in the “acute intermittent” and “mixed” forms of the disease.
Administration of the porphyria-inducing chemicals allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1, 4-dihydrocollidine (DDC) to rabbits, results in the development of marked elevation of serum cholesterol, total lipids and phospholipids. The liver total lipids, cholesterol lipid phosphorus and water concentrations remain unchanged, but the organ becomes greatly enlarged, reflecting active synthesis of new protoplasm. It is suggested that these compounds affect the lipoprotein transport of serum cholesterol in a manner inducing sequestration of the synthesized cholesterol in the plasma and preventing the accumulation of cholesterol in the liver cells hence, the activation of the hepatic feed-back control for cholesterol synthesis. The possi-
S
INCE
THE
FIRST
experimental
DESCRIPTlON
hepatic
porphyria
J. WATSON
C.
by
induced
S&mid
and
Schwartz’
of
an
by allvlisopropylacetylcarbamide
( SedormidB ), a number of other chemicals including allylisopropylacetamide ( AI A ),’ hexachlorobenzene,:’ 3,5-dicarbethoxy-1, 4dihydrocollidine (DDC ),-I and griseofulvin,” of porphyrins have recently piss
have been found
and
their
is due to increased
activity
of the liver.
porphyrogenic
embryo
in varying
shown that porphyrin
in the mitochondria that
to cause excessive
precursors,
chemicals
formation
degree.
overproduction
induced
of S-aminolevulinic Subsequently,
indllce
the
and
by DDC
acid
svnthetase
provided
of this
evidence
enzyme
in chick
liver cell culture.
Schwartzx first observed presence of disturbed lipid metabolism mental porphyria, i.e., increase of total lipid and the phospholipid rat liver ported
Urata’;
in guinea
(ALA)
Granicki
synthesis
and excretion
Granick
after
that
is incrrascd lationship
administration
the synthesis about between
twofold heme
From the, Department Afinnesrrtu.
of Sedormid.
of fatty
acids
and postulated and lipid
of Medicine,
Labbe
and
by the livers
Uniaersity
of
associates”
of AIA
the existence
metabolism
in experifraction ot later
porphyric
of some
type
rcrats
of rc-
in the liver. Minnesota
Aided by grants from the U.S.P.H.S.. cmd the John and Mnry seurch Fund. Rcceiwd for Imblkation Mar. 5, 1964.
Hospital, Briggs
Minneapolis, Porphyria
Hc-
691 METABOLISM,
VOI.. 13, No. 8 ( AUCXJST), 19fS
Gl-OUPS*
in
significant.
7N.S.
not
*Differences
=
the
of
Significance
initial
values
differences
,003
<.OOl
3.2x
3.75
B-A
P
in
1435
Sl3-UNl
in duplicate.
12 rabbits,
-c 209
final
not
values
statistically
the
<.OOl
<.OOl
4.83
1.69
Cholesterol t P
out
from
207
1890
131
i
1533
f
1976
-c 164
1535
k 138
1910
Final Gm.
rt 154
Initial Gill.
were
carried
t
Total Lipids
was
an average
Fasting
Fasting
C-A
Compared
Diet
Fasting
represents
determination
DDC
C
Each
AIA
B
figure
None
A
Each
Drug
GKXIP
Body Weight
and
<.OOl
groups
<.OOl
significant.
between
6.67
4.24
Lipid Phosphorus t P
6 males
t
B
standard
t
i
A
and
groups
<.OOl
and
<.OOl
p
5.6i
Fresh Weight
9.99
t
260
41.5
134
-c 78.9
332
k 80.3
2
Cholestwo1 mg./ 100 ml.
A.
N.S.
C and
N.S.7
P
0.00
Dry Weight
Data
2.21
t
Data Final
deviation.
309
990
231
8Sl
k 187
599
Total Lipids mg./ 100 ml.
Table S.-Statistical
k
1.91
5.46
k l.Oi
5.31
-C l.i4
5.62
Lipid Phosphorus mg./ 100 ml.
6 females,
19.8
25.0
56.5
59.9 i
i
350
123
386
k 117
2
24.9
63.i i
391 I176
Cholestwo1 mg./ 100 ml.
Total Lipids mg./ 100 ml.
Initial
SelXIIl
Table l.-Experimental
f
t
1.14
P
Liver
9.60
55.6
N.S.
N.S.
i
-c 9.20
67.2
I5.10
36.9
Fresh Weight Gm.
Total Lipids
1.96
2.93
13.2
k 2.11
10.0
k 1.69
6.69
Lipid Phosphorus mg./ 100 ml.
I 85
45.0
220
43.0
220
I .06
.I40
N.S.
N.S.
Ch&itrrol t P
i- 21.0
I
r
Dry Weight mg., 1 Gm. fr. wt.
i
i
i
13.7
34.8
24.8
55.8
10.6
40.5
Total Lipids mg./ 1 Gm. fr. wt.
.430
2.16
.860
2.40
.490
2.36
1.73
1.13
N.S.
N.S.
Lipid Phosphorus t P
=
t
2
Cholestwo1 mg./ 1 Gm. fr. wt.
1.18
.240
1.05
i
i
2.0
78.0
k 6.0
12.4
2 5.0
74.0
water content %
2.47
.711
N.S.
N.S.
water content t P
,170
.900
k ,320
i
Lipid Phosphorus mg./ 1 Gm. fr. wt.
IIYPERCHOLES’LXROLEMIAIN HEPAWC PORPHYRIA
It has now been found of AIA or DDC develop phospholipids. stimulate concomitant
that rabbits made porphyric marked elevation of serum
In addition,
synthesis
evidence
of protein
records
prtwwt
rt~pw?.
of patients
with
b~ATEHlAL
“;ctl libltutll“ w;~t~r tur
fur 4 days the intake
A group
after
cluratioll
~~lilllinwto differences in dietary
arrival
of the
between
treated
of 12 rabbits,
of
qro~q
~ng./Kg, Twelve NaCL givrn
12 animals, of crystalline
rabbits,
and
6 of each
control
in 0.85
per
Blood
wa:, just
cent
sex,
GIII. were
served
and
AlAt
the
was
Purina
‘rabbit
fasted
hut
was
considered
otherwise
~~ht,cl pellet>
permitted rssential
related
to
to variations
animals. give
a singly
NaCl)
claily
dose
subcutaneously,
wrrr given by gavagtd,
of 250
for
mg./Kg.
7 clays.
of
A second
a single daily dose of 250 for the same period of time.
as controls; 6 were and the remaining
given 1617 ml./Kg. of 0.85 6 were intuhated daily and
1 2 ho11rs after initiation of drawn from each animal by cardiac punrtllre. before the first dose of AIA or DDC, and again at the end of the 7 day were then c~sanguinatetl The liver was removed,
and
analysis
specimens from
of- :I of
fed
period of administration. The animals by \vctioning thcz inferior vena cava. t;iinc(l
basis
wcsre then
deprivation
6 males and 6 females, DDC suspended in water,
6 of each
the
Crystallinr
conccsotration
sex, were
solution daily, subcutaneously, 20 ml. of water.
fasting,
Food lipid
results
from a surve\
METHODS
1,300-2,200
liver
the
form
ethanol-water.
at the laboratory
arid
findings,
porphyria,
AND hot
experitnent.
in serum
( 1.5 per cent solution
AI.4
These
and data obtained
hepatic
L)Ix* MUS Icuystallized twice from :,?I uhtailWcl. \\:llitta rabhita of both hexes weighing
by the administration cholesterol and serum obtained that AIA and DDC
has been
by the liver.
study of liver lipid distribution
of the
693
\vere
taken
the Madder
for
lipid
of each
( wG j All wru~n halopIes
rabbit
and
under
gently
histologic
to he tcaatetl for
light ether :mctstht& blotted and weighctl
examination.
Urine
albumin
gluccw-,
and
was
oh-
porphohilirl-
0ge11
~)l~rrls”
and
total
were
serum
analyzed for total lipitls.~” total cholestcrol.~~ lipid hilirubin .I.? Scarmn protein fractionation hy elrctrophoresis
carric tl out in 6 AIA and 6 DDC animals Livt,r
lipids
were> estimated
according
thr. wa.\h
\pfacimen
In anotllcr (:tn.
Three
and
experiment received
were water
the
used
for
content
the
calculated lipids
were
daily
determination
was
of total
rabbits
AIA 250 mg./Kg.
was
at the beginning and at the end of the experiment. to the method of Folch et al.,‘” and alicptots of
amount
8 malt
phos-
of liver from
the
cholesterol”
dry
ant1
constant
wt,ight
approximately
2,000
present.15
Ilsecl,
each
subcutaneously,
weighing :md 3 were
given
250/mg./Kg.
crystalline. DDC suspended in water, by gavagc, daily, for 10 days. Two animals were rlacd as controls and all \\rere allowed food and water “at1 lihitmn.” Bloocl for serllln clurlc-strrol determinations was obtained fro111 each animal by cardiac puncture, at tilt. I~t?&lling
of the
experiment,
thc~rc~aftc~r for a period l’ht~ clinical :JOO patic>ntc
data with
LIP
in the hepatic
before
administration
to 36 clays. All blond present porphyria
strlcly
were
studied
of the
drug, and at regular intervals wercs drawn after a 12 hour fast.
samples obtained
over
the
from
paht
a survey
of the
recortls
of
22 years.
RESULTS Both lesterol
AIA
and
DDC
and phospholipids
“Eastman fConrtc,sy
Kodak Co., of Hoffmann
caused
regular
the effect
Rochester, La Roche
elevation
of DDC
N. Y. Inc., Nutley,
N. J.
of serum
being
more
total marked
lipids,
cho-
(tables
1
694
TADDEINI,
5oc
NORDSTROM
AND WATSON
0
I-
+ Control l
AIA
o DDC 0
400 0 .
0 .
8 r .-=
0 0
3oc
0
I-
.
0 & 2 0 6 5
.
.
0
l
2oc
00 0
. .
. I -
cn”
+
.
+
2 II
+ + . I oc
)-
++ +
+ + +
C)i
I
50
I
IO0
I
150
Liver cholesterol
I
200
I
250
I
300
I
350
in mg. %
Fig. l.- Relation between serum and liver cholesterol concentrations in AIA, DDC and control rabbits. and 2). The control animals showed a moderate degree of hyperlipemia and hypercholesterolemia as a consequence of fasting as previously observed.1s-18 Only 2 rabbits of the 24 in the experimental group failed to develop significant elevation of the serum cholesterol and had values falling within the range of the control group (fig. 1). Liver water content, total lipid, cholesterol and lipid phosphorus concentrations of the treated rabbits did not differ significantly from those of the control animals and did not parallel the serum levels of these substances (tables 1, 2 and fig. 1). H owever, the livers of the rabbits receiving AIA or DDC were considerably larger than those of the normal animals, conse-
HTPERCHOLESTEROLEhfIA
IN HEPATIC:
W5
PORPHYRIA
3OOr
RabbII
I
RobbIt
3
Robbtt
2
6Oy
c
20
Period of \\\\\\\\\\\\\\\\\q AIA
0 Fig. 2.-Time
Administration
250mg/
relationship
I I 12 Days
kg/day
4
8
of scrutn cholesterol
I 16
I
I 20
1
I 24
J
Ievcls in 3 AIA-treated
rahhits.
quently
the content of total liver lipid and its fractions were almost invariably higher in the treated than in the control group (tables 1 and a). It was also apparent that the considerable growth of the liver in the ALA and DDC animals, whether due to hypertrophy or cell multiplication, and in the absence of any histologic evidence of cirrhosis or glycogen infiltration. reflected new formation of liver cell protoplasm. This conclusion was supported by previous observations on the development of hepatomegaly with and normal concentration of ribo- and normal liver nitrogen concentration’” desoxy-ribonucleic acid*@.*’ in experimental porphyria, also by the finding of an increased protein and ribonucleic acid content in tissue culture cells grown in the presence of porphyria-inducing drugs.“’ The results summarized in figures 2. and 3 show that an appreciable elevation of serum cholesterol was already manifest on the second day of treatment and became progressively more marked. Discontinuation of the drugs resulted in a slow return to normal levels. A further rise of serum cholesterol concentration of chemical, Serum
observed
protein
administration from a mean
in the DDC-treated
may be related
to delayed
electrophoresis, of the
drugs,
performed revealed
rabbits
absorption
6 days after
in 12 animals
a slight
the last dose
in the intestinal increase
value of 0.83 Gm. per cent to a mean
before in the
tract. and
after
(Y globulins
of 1.03 Gm. per cent.
and
696
TADDEINI,
950 -
NORDSTROM
AND
WATSON
!
850 -
\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ‘\\ \ \ \ \ \ ‘. ‘\ Rabbit
5
I
0
4
8
12
16
20
24
28
32
36
Days Fig.
S.-Time
relationship of serum cholesterol levels in 3 DDC-treated rabbits.
of the /3 globulins from a mean of 0.83 Gm. per cent to a mean of 1.02 Gm. per cent. Serum bilirubin level remained unchanged in the porphyric rabbits and blood glucose and urea nitrogen were also unaffected. There was no glycosuria or albuminuria, but the test for PBG was invariably positive. From the review of the records of 300 patients with various forms of hepatic porphyria, 23 51 were found to have one or more cholesterol values recorded. In most instances cholesterol and cholesterol ester determinations had been made as part of the study of liver function and’ had not been prompted by clinical manifestations usually associated with hypercholesterolemia. The pertinent data are given in table 3. Because of differences in method, year of determination and other factors, an exact comparison with a control group has not been possible. Our data, however, seem to indicate that a significant elevation of serum cholesterol, even if not constantly present, is of frequent occurrence in the “acute intermittent” and “mixed” types.
HYPERCHOLESTEROLEMIA
IN HEPATIC
PORPHYHIA
697
The finding in the literature of a number of cases of hepatic porphyria. in which high serum cholesterol values were casually mentioned,“-‘” tends to corroborate the observation that hypercholesterolemia is at least relatively common in this group. DISCUSSION
The level of plasma cholesterol reflects the sum of cholesterol from the diet and that from endogenous synthesis in relation to the cholesterol rc‘moved from the plasma by catabolism and excretion. The possibility also exists that the plasma lipoproteins and their cholesterol may redistribute themselves between plasma and interstitial fluid or that the structure ot the lipoproteins may be altered. Sakakida and associates,‘” by means of cross-infusion experiments, have reported that in the stilbestrol-induced hypercholesterolemia of the rooster,“” ihc lipoprotein complex is altered in a manner causing sequestration of cholesterol in the plasma and preventing accumulation of cholesterol in the lilrer. As ;\ result the liver cholesterol concentration is normal, thus there is failure of the hepatic feed back control leading to further synthesis of cholesterol. From the present finding of an elevation of serum cholesterol accompanied bv normal liver cholesterol concentration in the AIA and DDC porphyric rabbits, it might be postulated that these drugs influence thus lipoprotein transport system in a similar manner. inducing sequestration of synthesized cholesterol in the plasma. It is noteworthy that AIA and DD<: porphyric rabbits develop marked enlargement of the liver. indicating 311 unusual ability of the liver cells to synthesize protein and suggesting the possibility of increased production of lipoprotein protein or the elaboration of a protein with unusual affinity for lipids, of the type postulated by Frantz.:” It seems not unlikely that AL4 and DDC may have a more direct influencr on the rate of hepatic cholesterol synthesis. Granick’ demonstrated that porphvrin overproduction, induced bv AIA. DDC and other chemicals in , I chick embryo liver cells, is due to an increase of ALA synthetase and suggested that these compounds activate a gene required for the synthesis of tha enzyme. It is conceivable that enzymes of cholesterol synthesis may be similarly affected with resulting accelerated cholesterol production by the liver. The porphyria-inducing chemicals, in fact, may have their primary effect on the genetic control of the rate of synthesis of different proteins bv tht liver. Although no attempt was made in the present study to correlate the degree of porphyrin overproduction with that of hypercholesterolemia in the individual animals, the observation that 2 different chemicals, quite unrelated, both induce elevation of serum cholesterol and increased porphyrin formation, suggests that the association is not coincidental. The additional finding of hypercholesterolemia in many cases of human hepatic porphyria also indicates a relationship between porphyrin and lipid metabolism ant 1 enhances the similarity of the experimental and human diseases. However. it
698
TADDEINI,
Table 3.-Clinical Patient
Age*
Sex
R. B.
31
hl. B. J. B. F. C. M. c. I>. E. C. C. c. I). c.
70 61 19 25
Al F F F F
35 28 26
F hf F
V. E. F. F.
36 34
F hi F F
1950 1962 1963
W. G.
27 39 40 46
RI M F
1948 1944 1956 1957 1957 1946 1950 1951 1946 1962 1963
Acute
J. H. S. H.
R. H. \V. J. RI. J. H. K. A. K. v. I\‘. E. M. W. N. R. N. N. 0. J. P. K. P. B. R. A. S. M. V. R. W.
53 31 32 32 17 43 30 24 ri4 55 27 32 25 56 23 25 55 39 29 36 67 21
F nr F 1’ RI F M
36.9 f
14.4 Mixed
E. A. ;Y%
J. C. T. G. G. H. W. J. A. R.
50 54 24 22 00 57 41 60 62
F M F F Al F M F
306 274 340 315 171 200 397 536 340 198 2’9 2.54 2.55 “S4 271 450 269 247 169 374 235 196 191 216 31X 237 384
1957 1959 1963 1952 1952 1952 1953 1950
hf F F F F M
Mean age -t S.D. =
250 434
1947 1953 1962 1962
M F F
Total Bilirubin mg./lOO ml
230 353 187 396 303 277 296 22”
1963 1951 1954 1942 1945 1953 1948 1956 1941 (8/5) 1941(8/21)
Alkaline Phosphatase units/100 ml.;
1.0 0.8 -
7 K.A. -
0.4 1.3 -
4.8 B
0.6 -
stage
of Diseasei:
A A A R
13 K.A. -
0.3 0.8 -
4.4 B -
0.5 0.7
5.3 K.A. -
0.4 1.4 -
6.ri K.A. -
0.2 -
-
6 K.A.
0.4 0.5 0.8 0.5 0.6 0.6 -
3.3 B 7.8 K.A. 4.0 B S.D.
R A A A A A A A A A R R R A R
11.5 K.A. -
k
R R R A A A A
-
0.7 -
hfean clmlesterol Hepatic
WATSON
Porphqrio
Intermittent
1956
AND
Data
Cholesterol mg./lOO ml.
Date*
NORDSTROM
=
R A A A A .4 A A R
273 *
75.2
Porphyrin
1960 1952 1961 1961
310 207 325 288
0.4 1.14 -
1948 1959 1955 1960 1962
285 “43 183 1115 177
0.4 0.5 1.6 0.3 0.8
6 K.A. 4.6 K.A. 6.5 K.A. 6 K.A. 9 K.A. 17 K.A.
R ? R R A R R R R
Table 3.--(Continued) _._~~.._
S.
Sex ~___
Age* ~_..___
Patient _~
R.
M
37
1950 (Oct.) 1950 (Nov.) 1957
ii4 Mran age i
S.D.
=
Total Bilirubin Cholesterol mg./lOlJ ml. mg./lOO ml. ________.
Date*
42.0
f
16.0
Cutuneu
stage uf Disease$
1.9 B 2.0 B 12.0 K.A.
A A 11
1.5 I .2 05
303 364 412
LIean cholestcTo1
Porphyriu
Alkaline Phosphatase units/100 ml.? ____~_~
t
S.D.
=
265 2
TUTdU
c:. A.
50
F
1955
137
c. B. H. C. J. c.
50 67 .58
hi M F
1950 1952 1955
192 184 155
1.3 0.3 0.7
6 K.A. -
E. H. H. H. E. M. \I. hl.
60 40 6-l ;i8
M M M I1
1950 1956 1963 1956
229 282 177 278
1.o 0.1 0.6 0.2
E. hl. L. 11. s. \‘.
61 40 48 10
F hl M
1959 1950 1964 1964
235 210 190 205
1.6 B 11.7 li.A. 14 K.A. 20.3 K.A. 20.5 K.A. -
Mean age zz S.D.
=
52.5
i
0.7
8 li.A. -
Mean cholesterol
9.95
63.5
i
S.D.
=
202
& 42.3
*At time of determinations. tB = $A =
Bodansky units; K.A. = King Armstrong Acute attack; R = Remission.
units.
would be difficult in the light of existing knowledge, to ascribe hypercholesterolemia in human porphyria to the same genetic abnormalitv I responsible for increased production of ALA, PBG and porphyrins. ACKNOWLEDGMENT Wt. acknowledge with gratitude preparation of this manuscript.
the advice
and snggeqtions
of Dr. Ivan
Frantz,
in the
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by
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-I-
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26.
study
Nicholas,
R. E.
of two families
H.,
having
Acute intermittent porphyria treated with chlorpromazine. Proc. Roy. Sot.
1927.
17. Fahrig, C., and Wacker, L.: Vergleichiiber den Untersuchungen ende des Blutserums bei Lipoidkomplex essentieller Hypertension, MuskelarSchwangerschaft und beit. Hunger, Nahrungsaufnahme. Klin. Wchnschr.
Watson,
porphyrie
in
in J.
Liver
Grotepass, W., Nyssen, R., and Van Bogaert, L.: Etude clinique, anatomique et chimique d’ un ens de
75:648.
1963. 16. Shope, R. E.: Sugar and cholesterol blood serum as related to fasting.
1957.
met porphyrinurie. Geneesk. 64:1233,
1951.
15. Zaki, G. F., Bandt, C., and Hofiauer, F. W.: Fatty cirrhosis in the rats. III.
liver
T. F., and Wang,
Eng. J. Med. 263:1205,
30:
tis-
95:221, F., Slater,
yria-some
293, 1945. 14. Folch, J,, Arcoli, I., Lees, M., Meath, J. A., and Le Baron, F. N.: Preparation
Y.:
Bioph.
C. J.: The quanof serum bilireference to the an d chloroform-
by
Sewell. M.: Oxidative metabolism of tissue culture cells in the presence of porphyria-inducing drugs. Amrch.Bioch.
Y.: The of phos-
66:375,
Med.
22.
D.
23.
and Subbarow, determination
porphyrins
mental hepatic porphyria. Bioph. Acta 68:100, 1963. Cowger, M. L., Labbe, R.
Chem.
1952.
12. Fiske, C. H., calorimetric
Biol.
demonstra-
J. Biol.
to
B. L., and of porpho-
De Matteis,
1955.
11. Abell, L. L., Levy, B. B., Brodie, B. B., and Kendall, F. E.: A simplified method for the estimation of total
1955.
21.
in blood
213:69,
217:263,
homogenates of rats with experimental hepatic porphyria. Proc. Sot. Esper.
porphyria.
Bioch.
Chem.
AND WATSON
Merchante, A., Wajchenberg, Schwartz, S.: Conversion bilinogen
F. I.: He:rle and fatty acid bio-
synthesis
NORDSTROM
29.
Med. 55:799, 1962. Sakakida, H., Shediac, C. C., and Siperstein, M. D.: The effect of endogenous cholesterol on the and exogenous feedback
control
of
cholesterol
syn-
thesis. J. Clin. Invest. 42:1521, 1963. I. L., and 30. Lorens, F. W., Chaikoff, Entenmen, C.: The endocrine control of lipid metabolism in the bird. II. The effect of estrin on the blood lipids of the immature domestic fowl. J. Biol. Chem. 126:763, 1938. 31. Fran@ I. D., Jr.: Cholesterol metabolism. Minnesota Med. 38:779, 1955.
IIPER(:IIOLESTEROLEhfIA
IN HEPATIC
70 1
PORPHYRIA
Luigi Tnddeini, M.D., Research Fellou~ in Medicine, Trainee irr Hematology, U.S.P.H.S.. Unioersity of Minnesota Me&cd School, Minneapolis. Minn. Krrrct~I,. Nordstrom, B.S., Research Technician, Unicersity Minnesota Medim School, Minnenpolis, Mim.
of
(:. J. Watson, M.D., Ph.D.. Professor of Neckzinc, Unicersity of Minnesota Medical Srhool, Minneapolis. Minn.
TADDEINI, KAREN L. NORDSTROMAND
bility also exists that AIA and DDC enhances the production of one or more enzymes of cholesterol synthesis, in a way analogous to what Granick and Urata have demonstrated to occur for the enzyme S-aminolevulinic acid synthetase of the porphyrin synthetic pathway. The chemicals, in fact, may have their primary effect on the genetic control of the rate of synthesis of different proteins by the liver. A review of the records of 51 patients with hepatic porphyria, in whom serum cholesterol determinations were recorded, reveals that hypercholesterolemia, although inconstant, is of common occurrence in the “acute intermittent” and “mixed” forms of the disease.
Administration of the porphyria-inducing chemicals allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1, 4-dihydrocollidine (DDC) to rabbits, results in the development of marked elevation of serum cholesterol, total lipids and phospholipids. The liver total lipids, cholesterol lipid phosphorus and water concentrations remain unchanged, but the organ becomes greatly enlarged, reflecting active synthesis of new protoplasm. It is suggested that these compounds affect the lipoprotein transport of serum cholesterol in a manner inducing sequestration of the synthesized cholesterol in the plasma and preventing the accumulation of cholesterol in the liver cells hence, the activation of the hepatic feed-back control for cholesterol synthesis. The possi-
S
INCE
THE
FIRST
experimental
DESCRIPTlON
hepatic
porphyria
J. WATSON
C.
by
induced
S&mid
and
Schwartz’
of
an
by allvlisopropylacetylcarbamide
( SedormidB ), a number of other chemicals including allylisopropylacetamide ( AI A ),’ hexachlorobenzene,:’ 3,5-dicarbethoxy-1, 4dihydrocollidine (DDC ),-I and griseofulvin,” of porphyrins have recently piss
have been found
and
their
is due to increased
activity
of the liver.
porphyrogenic
embryo
in varying
shown that porphyrin
in the mitochondria that
to cause excessive
precursors,
chemicals
formation
degree.
overproduction
induced
of S-aminolevulinic Subsequently,
indllce
the
and
by DDC
acid
svnthetase
provided
of this
evidence
enzyme
in chick
liver cell culture.
Schwartzx first observed presence of disturbed lipid metabolism mental porphyria, i.e., increase of total lipid and the phospholipid rat liver ported
Urata’;
in guinea
(ALA)
Granicki
synthesis
and excretion
Granick
after
that
is incrrascd lationship
administration
the synthesis about between
twofold heme
From the, Department Afinnesrrtu.
of Sedormid.
of fatty
acids
and postulated and lipid
of Medicine,
Labbe
and
by the livers
Uniaersity
of
associates”
of AIA
the existence
metabolism
in experifraction ot later
porphyric
of some
type
rcrats
of rc-
in the liver. Minnesota
Aided by grants from the U.S.P.H.S.. cmd the John and Mnry seurch Fund. Rcceiwd for Imblkation Mar. 5, 1964.
Hospital, Briggs
Minneapolis, Porphyria
Hc-
691 METABOLISM,
VOI.. 13, No. 8 ( AUCXJST), 19fS
Gl-OUPS*
in
significant.
7N.S.
not
*Differences
=
the
of
Significance
initial
values
differences
,003
<.OOl
3.2x
3.75
B-A
P
in
1435
Sl3-UNl
in duplicate.
12 rabbits,
-c 209
final
not
values
statistically
the
<.OOl
<.OOl
4.83
1.69
Cholesterol t P
out
from
207
1890
131
i
1533
f
1976
-c 164
1535
k 138
1910
Final Gm.
rt 154
Initial Gill.
were
carried
t
Total Lipids
was
an average
Fasting
Fasting
C-A
Compared
Diet
Fasting
represents
determination
DDC
C
Each
AIA
B
figure
None
A
Each
Drug
GKXIP
Body Weight
and
<.OOl
groups
<.OOl
significant.
between
6.67
4.24
Lipid Phosphorus t P
6 males
t
B
standard
t
i
A
and
groups
<.OOl
and
<.OOl
p
5.6i
Fresh Weight
9.99
t
260
41.5
134
-c 78.9
332
k 80.3
2
Cholestwo1 mg./ 100 ml.
A.
N.S.
C and
N.S.7
P
0.00
Dry Weight
Data
2.21
t
Data Final
deviation.
309
990
231
8Sl
k 187
599
Total Lipids mg./ 100 ml.
Table S.-Statistical
k
1.91
5.46
k l.Oi
5.31
-C l.i4
5.62
Lipid Phosphorus mg./ 100 ml.
6 females,
19.8
25.0
56.5
59.9 i
i
350
123
386
k 117
2
24.9
63.i i
391 I176
Cholestwo1 mg./ 100 ml.
Total Lipids mg./ 100 ml.
Initial
SelXIIl
Table l.-Experimental
f
t
1.14
P
Liver
9.60
55.6
N.S.
N.S.
i
-c 9.20
67.2
I5.10
36.9
Fresh Weight Gm.
Total Lipids
1.96
2.93
13.2
k 2.11
10.0
k 1.69
6.69
Lipid Phosphorus mg./ 100 ml.
I 85
45.0
220
43.0
220
I .06
.I40
N.S.
N.S.
Ch&itrrol t P
i- 21.0
I
r
Dry Weight mg., 1 Gm. fr. wt.
i
i
i
13.7
34.8
24.8
55.8
10.6
40.5
Total Lipids mg./ 1 Gm. fr. wt.
.430
2.16
.860
2.40
.490
2.36
1.73
1.13
N.S.
N.S.
Lipid Phosphorus t P
=
t
2
Cholestwo1 mg./ 1 Gm. fr. wt.
1.18
.240
1.05
i
i
2.0
78.0
k 6.0
12.4
2 5.0
74.0
water content %
2.47
.711
N.S.
N.S.
water content t P
,170
.900
k ,320
i
Lipid Phosphorus mg./ 1 Gm. fr. wt.
IIYPERCHOLES’LXROLEMIAIN HEPAWC PORPHYRIA
It has now been found of AIA or DDC develop phospholipids. stimulate concomitant
that rabbits made porphyric marked elevation of serum
In addition,
synthesis
evidence
of protein
records
prtwwt
rt~pw?.
of patients
with
b~ATEHlAL
“;ctl libltutll“ w;~t~r tur
fur 4 days the intake
A group
after
cluratioll
~~lilllinwto differences in dietary
arrival
of the
between
treated
of 12 rabbits,
of
qro~q
~ng./Kg, Twelve NaCL givrn
12 animals, of crystalline
rabbits,
and
6 of each
control
in 0.85
per
Blood
wa:, just
cent
sex,
GIII. were
served
and
AlAt
the
was
Purina
‘rabbit
fasted
hut
was
considered
otherwise
~~ht,cl pellet>
permitted rssential
related
to
to variations
animals. give
a singly
NaCl)
claily
dose
subcutaneously,
wrrr given by gavagtd,
of 250
for
mg./Kg.
7 clays.
of
A second
a single daily dose of 250 for the same period of time.
as controls; 6 were and the remaining
given 1617 ml./Kg. of 0.85 6 were intuhated daily and
1 2 ho11rs after initiation of drawn from each animal by cardiac punrtllre. before the first dose of AIA or DDC, and again at the end of the 7 day were then c~sanguinatetl The liver was removed,
and
analysis
specimens from
of- :I of
fed
period of administration. The animals by \vctioning thcz inferior vena cava. t;iinc(l
basis
wcsre then
deprivation
6 males and 6 females, DDC suspended in water,
6 of each
the
Crystallinr
conccsotration
sex, were
solution daily, subcutaneously, 20 ml. of water.
fasting,
Food lipid
results
from a surve\
METHODS
1,300-2,200
liver
the
form
ethanol-water.
at the laboratory
arid
findings,
porphyria,
AND hot
experitnent.
in serum
( 1.5 per cent solution
AI.4
These
and data obtained
hepatic
L)Ix* MUS Icuystallized twice from :,?I uhtailWcl. \\:llitta rabhita of both hexes weighing
by the administration cholesterol and serum obtained that AIA and DDC
has been
by the liver.
study of liver lipid distribution
of the
693
\vere
taken
the Madder
for
lipid
of each
( wG j All wru~n halopIes
rabbit
and
under
gently
histologic
to he tcaatetl for
light ether :mctstht& blotted and weighctl
examination.
Urine
albumin
gluccw-,
and
was
oh-
porphohilirl-
0ge11
~)l~rrls”
and
total
were
serum
analyzed for total lipitls.~” total cholestcrol.~~ lipid hilirubin .I.? Scarmn protein fractionation hy elrctrophoresis
carric tl out in 6 AIA and 6 DDC animals Livt,r
lipids
were> estimated
according
thr. wa.\h
\pfacimen
In anotllcr (:tn.
Three
and
experiment received
were water
the
used
for
content
the
calculated lipids
were
daily
determination
was
of total
rabbits
AIA 250 mg./Kg.
was
at the beginning and at the end of the experiment. to the method of Folch et al.,‘” and alicptots of
amount
8 malt
phos-
of liver from
the
cholesterol”
dry
ant1
constant
wt,ight
approximately
2,000
present.15
Ilsecl,
each
subcutaneously,
weighing :md 3 were
given
250/mg./Kg.
crystalline. DDC suspended in water, by gavagc, daily, for 10 days. Two animals were rlacd as controls and all \\rere allowed food and water “at1 lihitmn.” Bloocl for serllln clurlc-strrol determinations was obtained fro111 each animal by cardiac puncture, at tilt. I~t?&lling
of the
experiment,
thc~rc~aftc~r for a period l’ht~ clinical :JOO patic>ntc
data with
LIP
in the hepatic
before
administration
to 36 clays. All blond present porphyria
strlcly
were
studied
of the
drug, and at regular intervals wercs drawn after a 12 hour fast.
samples obtained
over
the
from
paht
a survey
of the
recortls
of
22 years.
RESULTS Both lesterol
AIA
and
DDC
and phospholipids
“Eastman fConrtc,sy
Kodak Co., of Hoffmann
caused
regular
the effect
Rochester, La Roche
elevation
of DDC
N. Y. Inc., Nutley,
N. J.
of serum
being
more
total marked
lipids,
cho-
(tables
1
694
TADDEINI,
5oc
NORDSTROM
AND WATSON
0
I-
+ Control l
AIA
o DDC 0
400 0 .
0 .
8 r .-=
0 0
3oc
0
I-
.
0 & 2 0 6 5
.
.
0
l
2oc
00 0
. .
. I -
cn”
+
.
+
2 II
+ + . I oc
)-
++ +
+ + +
C)i
I
50
I
IO0
I
150
Liver cholesterol
I
200
I
250
I
300
I
350
in mg. %
Fig. l.- Relation between serum and liver cholesterol concentrations in AIA, DDC and control rabbits. and 2). The control animals showed a moderate degree of hyperlipemia and hypercholesterolemia as a consequence of fasting as previously observed.1s-18 Only 2 rabbits of the 24 in the experimental group failed to develop significant elevation of the serum cholesterol and had values falling within the range of the control group (fig. 1). Liver water content, total lipid, cholesterol and lipid phosphorus concentrations of the treated rabbits did not differ significantly from those of the control animals and did not parallel the serum levels of these substances (tables 1, 2 and fig. 1). H owever, the livers of the rabbits receiving AIA or DDC were considerably larger than those of the normal animals, conse-
HTPERCHOLESTEROLEhfIA
IN HEPATIC:
W5
PORPHYRIA
3OOr
RabbII
I
RobbIt
3
Robbtt
2
6Oy
c
20
Period of \\\\\\\\\\\\\\\\\q AIA
0 Fig. 2.-Time
Administration
250mg/
relationship
I I 12 Days
kg/day
4
8
of scrutn cholesterol
I 16
I
I 20
1
I 24
J
Ievcls in 3 AIA-treated
rahhits.
quently
the content of total liver lipid and its fractions were almost invariably higher in the treated than in the control group (tables 1 and a). It was also apparent that the considerable growth of the liver in the ALA and DDC animals, whether due to hypertrophy or cell multiplication, and in the absence of any histologic evidence of cirrhosis or glycogen infiltration. reflected new formation of liver cell protoplasm. This conclusion was supported by previous observations on the development of hepatomegaly with and normal concentration of ribo- and normal liver nitrogen concentration’” desoxy-ribonucleic acid*@.*’ in experimental porphyria, also by the finding of an increased protein and ribonucleic acid content in tissue culture cells grown in the presence of porphyria-inducing drugs.“’ The results summarized in figures 2. and 3 show that an appreciable elevation of serum cholesterol was already manifest on the second day of treatment and became progressively more marked. Discontinuation of the drugs resulted in a slow return to normal levels. A further rise of serum cholesterol concentration of chemical, Serum
observed
protein
administration from a mean
in the DDC-treated
may be related
to delayed
electrophoresis, of the
drugs,
performed revealed
rabbits
absorption
6 days after
in 12 animals
a slight
the last dose
in the intestinal increase
value of 0.83 Gm. per cent to a mean
before in the
tract. and
after
(Y globulins
of 1.03 Gm. per cent.
and
696
TADDEINI,
950 -
NORDSTROM
AND
WATSON
!
850 -
\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ‘\\ \ \ \ \ \ ‘. ‘\ Rabbit
5
I
0
4
8
12
16
20
24
28
32
36
Days Fig.
S.-Time
relationship of serum cholesterol levels in 3 DDC-treated rabbits.
of the /3 globulins from a mean of 0.83 Gm. per cent to a mean of 1.02 Gm. per cent. Serum bilirubin level remained unchanged in the porphyric rabbits and blood glucose and urea nitrogen were also unaffected. There was no glycosuria or albuminuria, but the test for PBG was invariably positive. From the review of the records of 300 patients with various forms of hepatic porphyria, 23 51 were found to have one or more cholesterol values recorded. In most instances cholesterol and cholesterol ester determinations had been made as part of the study of liver function and’ had not been prompted by clinical manifestations usually associated with hypercholesterolemia. The pertinent data are given in table 3. Because of differences in method, year of determination and other factors, an exact comparison with a control group has not been possible. Our data, however, seem to indicate that a significant elevation of serum cholesterol, even if not constantly present, is of frequent occurrence in the “acute intermittent” and “mixed” types.
HYPERCHOLESTEROLEMIA
IN HEPATIC
PORPHYHIA
697
The finding in the literature of a number of cases of hepatic porphyria. in which high serum cholesterol values were casually mentioned,“-‘” tends to corroborate the observation that hypercholesterolemia is at least relatively common in this group. DISCUSSION
The level of plasma cholesterol reflects the sum of cholesterol from the diet and that from endogenous synthesis in relation to the cholesterol rc‘moved from the plasma by catabolism and excretion. The possibility also exists that the plasma lipoproteins and their cholesterol may redistribute themselves between plasma and interstitial fluid or that the structure ot the lipoproteins may be altered. Sakakida and associates,‘” by means of cross-infusion experiments, have reported that in the stilbestrol-induced hypercholesterolemia of the rooster,“” ihc lipoprotein complex is altered in a manner causing sequestration of cholesterol in the plasma and preventing accumulation of cholesterol in the lilrer. As ;\ result the liver cholesterol concentration is normal, thus there is failure of the hepatic feed back control leading to further synthesis of cholesterol. From the present finding of an elevation of serum cholesterol accompanied bv normal liver cholesterol concentration in the AIA and DDC porphyric rabbits, it might be postulated that these drugs influence thus lipoprotein transport system in a similar manner. inducing sequestration of synthesized cholesterol in the plasma. It is noteworthy that AIA and DD<: porphyric rabbits develop marked enlargement of the liver. indicating 311 unusual ability of the liver cells to synthesize protein and suggesting the possibility of increased production of lipoprotein protein or the elaboration of a protein with unusual affinity for lipids, of the type postulated by Frantz.:” It seems not unlikely that AL4 and DDC may have a more direct influencr on the rate of hepatic cholesterol synthesis. Granick’ demonstrated that porphvrin overproduction, induced bv AIA. DDC and other chemicals in , I chick embryo liver cells, is due to an increase of ALA synthetase and suggested that these compounds activate a gene required for the synthesis of tha enzyme. It is conceivable that enzymes of cholesterol synthesis may be similarly affected with resulting accelerated cholesterol production by the liver. The porphyria-inducing chemicals, in fact, may have their primary effect on the genetic control of the rate of synthesis of different proteins bv tht liver. Although no attempt was made in the present study to correlate the degree of porphyrin overproduction with that of hypercholesterolemia in the individual animals, the observation that 2 different chemicals, quite unrelated, both induce elevation of serum cholesterol and increased porphyrin formation, suggests that the association is not coincidental. The additional finding of hypercholesterolemia in many cases of human hepatic porphyria also indicates a relationship between porphyrin and lipid metabolism ant 1 enhances the similarity of the experimental and human diseases. However. it
698
TADDEINI,
Table 3.-Clinical Patient
Age*
Sex
R. B.
31
hl. B. J. B. F. C. M. c. I>. E. C. C. c. I). c.
70 61 19 25
Al F F F F
35 28 26
F hf F
V. E. F. F.
36 34
F hi F F
1950 1962 1963
W. G.
27 39 40 46
RI M F
1948 1944 1956 1957 1957 1946 1950 1951 1946 1962 1963
Acute
J. H. S. H.
R. H. \V. J. RI. J. H. K. A. K. v. I\‘. E. M. W. N. R. N. N. 0. J. P. K. P. B. R. A. S. M. V. R. W.
53 31 32 32 17 43 30 24 ri4 55 27 32 25 56 23 25 55 39 29 36 67 21
F nr F 1’ RI F M
36.9 f
14.4 Mixed
E. A. ;Y%
J. C. T. G. G. H. W. J. A. R.
50 54 24 22 00 57 41 60 62
F M F F Al F M F
306 274 340 315 171 200 397 536 340 198 2’9 2.54 2.55 “S4 271 450 269 247 169 374 235 196 191 216 31X 237 384
1957 1959 1963 1952 1952 1952 1953 1950
hf F F F F M
Mean age -t S.D. =
250 434
1947 1953 1962 1962
M F F
Total Bilirubin mg./lOO ml
230 353 187 396 303 277 296 22”
1963 1951 1954 1942 1945 1953 1948 1956 1941 (8/5) 1941(8/21)
Alkaline Phosphatase units/100 ml.;
1.0 0.8 -
7 K.A. -
0.4 1.3 -
4.8 B
0.6 -
stage
of Diseasei:
A A A R
13 K.A. -
0.3 0.8 -
4.4 B -
0.5 0.7
5.3 K.A. -
0.4 1.4 -
6.ri K.A. -
0.2 -
-
6 K.A.
0.4 0.5 0.8 0.5 0.6 0.6 -
3.3 B 7.8 K.A. 4.0 B S.D.
R A A A A A A A A A R R R A R
11.5 K.A. -
k
R R R A A A A
-
0.7 -
hfean clmlesterol Hepatic
WATSON
Porphqrio
Intermittent
1956
AND
Data
Cholesterol mg./lOO ml.
Date*
NORDSTROM
=
R A A A A .4 A A R
273 *
75.2
Porphyrin
1960 1952 1961 1961
310 207 325 288
0.4 1.14 -
1948 1959 1955 1960 1962
285 “43 183 1115 177
0.4 0.5 1.6 0.3 0.8
6 K.A. 4.6 K.A. 6.5 K.A. 6 K.A. 9 K.A. 17 K.A.
R ? R R A R R R R
Table 3.--(Continued) _._~~.._
S.
Sex ~___
Age* ~_..___
Patient _~
R.
M
37
1950 (Oct.) 1950 (Nov.) 1957
ii4 Mran age i
S.D.
=
Total Bilirubin Cholesterol mg./lOlJ ml. mg./lOO ml. ________.
Date*
42.0
f
16.0
Cutuneu
stage uf Disease$
1.9 B 2.0 B 12.0 K.A.
A A 11
1.5 I .2 05
303 364 412
LIean cholestcTo1
Porphyriu
Alkaline Phosphatase units/100 ml.? ____~_~
t
S.D.
=
265 2
TUTdU
c:. A.
50
F
1955
137
c. B. H. C. J. c.
50 67 .58
hi M F
1950 1952 1955
192 184 155
1.3 0.3 0.7
6 K.A. -
E. H. H. H. E. M. \I. hl.
60 40 6-l ;i8
M M M I1
1950 1956 1963 1956
229 282 177 278
1.o 0.1 0.6 0.2
E. hl. L. 11. s. \‘.
61 40 48 10
F hl M
1959 1950 1964 1964
235 210 190 205
1.6 B 11.7 li.A. 14 K.A. 20.3 K.A. 20.5 K.A. -
Mean age zz S.D.
=
52.5
i
0.7
8 li.A. -
Mean cholesterol
9.95
63.5
i
S.D.
=
202
& 42.3
*At time of determinations. tB = $A =
Bodansky units; K.A. = King Armstrong Acute attack; R = Remission.
units.
would be difficult in the light of existing knowledge, to ascribe hypercholesterolemia in human porphyria to the same genetic abnormalitv I responsible for increased production of ALA, PBG and porphyrins. ACKNOWLEDGMENT Wt. acknowledge with gratitude preparation of this manuscript.
the advice
and snggeqtions
of Dr. Ivan
Frantz,
in the
REFERENCES I. Schmid,
R.,
mental produced Exptl.
and Schwartz,
porphyria-III Biol.
S.:
Esperi-
Hepatic
type
Sedormid.
Proc.
Sot.
Med. 81:685,
1952.
by
2. C:oldbcrg, A., Rimington, C., and Fenton, J. C. B.: Experimentally produced porphyria in animals. Proc. Roy. Sot.
porphyria
caused
Ac-
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IIPER(:IIOLESTEROLEhfIA
IN HEPATIC
70 1
PORPHYRIA
Luigi Tnddeini, M.D., Research Fellou~ in Medicine, Trainee irr Hematology, U.S.P.H.S.. Unioersity of Minnesota Me&cd School, Minneapolis. Minn. Krrrct~I,. Nordstrom, B.S., Research Technician, Unicersity Minnesota Medim School, Minnenpolis, Mim.
of
(:. J. Watson, M.D., Ph.D.. Professor of Neckzinc, Unicersity of Minnesota Medical Srhool, Minneapolis. Minn.