EXPERIMENTAL
The
AND
Effect
hlOLECULAR
of Pyridoxine
J. C. *Department and fDunn
PATHOLOGY
of Pathology, University Nutritional Laboratory, Milton July
( 1978)
Deficiency on Lysyl Oxidase in the Chick
Activity
D. R. FRASER,~ AND C. I. LEVENE”~~
MuRRA%,*J
Received
28, 301-308
of Cambridge, Tennis Court Road, Cambridge University of Cambridge and Medical Research Road, Cambridge, England
6, 1977,
and in revised
form
October
CB2 IQP Council,
17, 1977
The activity of lysyl oxidase, the enzyme responsible for the production of the precursor of the crosslinks of collagen and elastin, was measured in the aortas and epiphyseal cartilage of chicks raised on pyridoxine-deficient and control diets. Lysyl oxidase activity was diminished in both tissues in the deficient chicks, although the activity declined more slowly in the aorta. Deficient chicks given a large dose of pyridoxine 14 hr before sacrifice showed a marked increase in lysyl oxidase levels in both tissues. It is concluded that a pyridoxine derivative as well as copper are essential cofactors for lysyl oxidase. INTRODUCTION
LysyI oxidase is the enzyme responsibIe for the oxidation of specific lysine and hydroxylysine residues in collagen and elastin to form the aldehyde precursors of the crosslinks in these important structural proteins. (For reviews see Bailey et al., 1974; Gallop and Paz, 1975; Tanzer, 1976). Lysyl oxidase therefore has a crucial role in determining the structural integrity of connective tissues. Crosslink defects of elastin, resulting in aortic rupture, have been demonstrated in copper-deficient animals (O’Dell et al., 1961; Shields et al., 1962). In addition to these defects, a pronounced decrease in plasma amine oxidase activity was also found in copper-deficient swine (Blaschko et al., 1965). Page and Benditt (1967) suggested that lysyl oxidase might belong to that group of amine oxidases which are known to be copper dependent and probabIy also pyridoxal phosphate dependent (Yasunobu and Yamada, 1963; Blaschko and Buffoni, 1965). Hill and Kim (1967) found that, in addition to a reduction in the amine oxidase activity of aorta from pyridoxine-deficient chicks, there was a corresponding reduction in the conversion of lysine to the crosslinks desmosine and isodesmosine. These authors therefore suggested that lysyl oxidase contains a pyridoxine derivative and that pyridoxine deficiency has the same effect upon elastin biosynthesis as copper deficiency. Starcher (1969) confirmed a reduction in the synthesis of crosslinks in elastin of pyridoxine-deficient chicks but, unlike Hill and Kim (1967), could find no change in total elastin. Because these earlier studies did 1 J.C.M. was the recipient of an MRC Research Studentship while 2 C.I.L. is a member of the External Scientific Staff of the Medical
doing this work. Research Council.
301 0014-4800/78/0283-0301$02.00/O All
Copyright 0 1978 rights of reproduction
by Academic Press, Inc. in any form reserved.
302
MURRAY,
FRASER,
AND
TABLE
LEVENE
I
Amormt (g/kg of die(.)
Q Vit.amin mi.xt.ure (lug/kg of diet,) : riboflavin, 15; thinmine hydrochloride, 15; calcium pantothenate, 20; folic acid, 6 ; hiotin, 0.6 ; cyanocohalmk, 0.02 ; nicotinxmide, 50; vitamin A acetate, 120 ; DL-wtocopheryl acetate 500 ; menaphthone, 40. Vitamin D given orally, .5 pg on arrival. b Brigg’s salt mixture (A. B. Cos, Brighton, Sussex, UK) : (g/kg of diet) ; CaCOs, 12.3; CnHPO+ 19.5; NazHPOI, 11.1; KCI, 12.3; RlgHOa, 4.2; RInS04, 0.27; ferric citrate, 0.261; ZnC03, 0.045; CuSO,, 0.0225; KI03, 0.0015.
not directly estimate the activity of lysyl osidase, we have investigated in the chick the possible influence of pyridosine deficiency on this enzyme using the specific tritium release assay of Pinnell and Martin (1968) in view of the high substrate specificity of the enzyme ( Siegel et al., 1970); we also examined the effect of pyridoxine deficiency on comlective tissue morphology. MATERIALS One-day-old maIe chicks of Light Sussex/Rhode Island Red stock were obtained from the National Institute for Research in Dairying (Shinfield, Berkshire, UK). L-[4,5-3H]lysine monohydrochloride (sp act., 8.7 Ci/mmole) was purchased from the Radiochemical Centre ( Amersham, Buckinghamshire, UK). h4ETHODS Chicks were kept in heated brooder cages supplied with drinking water. Experimental groups were raised on a pyridosine-deficient diet (Table I) modified after Gries and Scott (1972) whiIe control groups were fed the same diet supplemented with pyridoxine hydrochloride (9 mg of pyridoxine/kg of diet). In the first two experiments, control and deficient groups were fed the diets ad Zibitum. In a third experiment the diet for the control group was restricted to give the same growth rate as the deficient chicks. In this latter experiment oral doses of 150 pg of pyridosine were given on Day 11 to two deficient chicks which were then killed 14 hr later. At the end of each experiment blood was collected by jugular venesection and the serum was stored at -20°C. Aortas and epiphyseal cartilage from the leg bones were removed and frozen prior to measurement of Iysyl oxidase activity. Serum filutamntc-oxalonrctate-tm~~sarninnse n,Psq. Serum glutamate-oxaloacetate-transaminase (GOT) activity was estimated by the method of Karmen ( 1955). Serum samples were incubated in the presence and absence of added pyridoxal-S-phosphate (5 x 1O-5 iJ4) at 25°C for 20 min before assaying. The
PYRIDOXINE
DEFICIENCY
AND CHICK
LYSYL
OXIDASE
303
ratio of the activities of the serum with and without added pyridoxal-S-phosphate is the activation coefficient, aGoT,which can be used as an index of pyridoxine status (Stanulovic et al., 1967). Preparation of substrate for lysyl ox&se assay. Insoluble substrate from chick embryo aorta IabeIed in vitro with L-[4,5-3H]Iysine was prepared essentially by the method of Pinnell and Martin ( 1968). Aortas from 36 chick embryos at 17 days of development were dissected and incubated at 37°C for 8 hr in 10 ml of Krebs-Ringer buffer (Manning and Meister, 1966) supplemented with [L-45 3H Jlysine monohydrochloride (25 &i/ml), p-aminopropionitrile fumarate (50 pg/ml), ascorbic acid (50 rg/ml), benzylpenicillin ( 100 U/ml) and streptomycin sulfate (100 U/ml). After incubation the medium was decanted, the aortas were washed twice with deionized water, then lyophilized, and stored at -20°C until required. To prepare the substrate for lysyl oxidase assays, labeled aortas were suspended in phosphate-buffered saline (PBS; 0.1 M NaH,P04-0.15 M NaCI, pH 7.7) and homogenized in a ground glass homogenizer. The resulting suspension was centrifuged at 17,OOOgfor 10 min at room temperature, resuspended in PBS, and boiled for 10 min to destroy endogenous lysyl oxidase activity. After centrifuging once more, the pellet was suspended in PBS to give approximately 100,000 cpm of tritium/0.5 ml. Extraction of lysyl oxidase. Although some lysyl oxidase activity can be extracted from chick tissues with PBS, the majority is rendered soIubIe onIy if concentrated urea is present in the buffer (Harris et al., 1974; Narayanan et al., 1974). In the experiments on epiphyseal cartilage and aorta, the salt- and the urea-soluble enzyme activities were assayed separately. Tissue from each group of chicks was pooIed and homogenized in ice-coId PBS (2 ml/g) with a SiIverson homogenizer and then centrifuged at 20,OOOgfor 20 min. The supernatant was retained and the process was repeated. The two supernatants were combined and used as a source of soluble lysyl oxidase. The residue was then twice extracted with 4 M urea-O.05 M Tris-HCI (pH 7.5) at room temperature in a similar manner. The supernatants were combined and used as a source of ureasoluble lysyl oxidase activity. Assay of tysy’l oxida.se activity. Before assaying, all urea extracts were dialyzed exhaustively against PBS at 4°C. Enzyme preparations together with 0.5 ml of labeled substrate were incubated in stoppered tubes with shaking at 37°C for 6 hr. Total volume for each assay was 1.5 ml. The reaction was terminated by adding 0.2 ml of 50% trichloroacetic acid and the tritiated water was obtained by vacuum distillation (Hutton et al., 1966). Radioactivity was measured using Kennedy’s scintillant ( 1969) with a 30% counting efficiency. The lysyl oxidase activity is given in units where 1 unit (U) corresponds to the release of 100 cpm of tritium/6-hr incubation/lOO,OOO cpm of substrate. Total enzyme activity is expressed as units per gram (wet weight) of tissue. All assays were done in duplicate. Preparation of sections for examination by light microscopy. A broad sample of organs and tissues collected from control and deficient chicks were placed immediately after dissection into 10% formal saline; particular attention was paid to tissues containing much elastin or collagen. Paraffin sections were prepared and stained with Harris’ hematoxylin and eosin and others with Weigert’s elastin stain, followed by iron hematoxylin counterstain and Curtis’ substitute for Van Gieson.
304
MURRAY,
FRASER,
AND
TABLE Serum
(;lutamate-Oxaloacetate-Tra~lsamillase Pyridoxine-ljeficient Chick
LEVENE
II and Cont.rol
weight
GOT
(GOT) Levels Chicks activity0
in S-I jay
ol-GOT*
k) Expt
II
Ad libitum cant rol Pyridoxine-deficient Expt III Rest.ricted diet. aIntro Pyridoxine-deficient
70.1 * 52.9 f
X;?’ 3.8
X0.6 * 40.4 f
5.5 3.7
I .04 f I .47 f
0.02 0.09”
.X.8 * 1.6 49.5 f 2.3
86.3 * 56.8 f
2.2 2.X
1.03 3z 0.0:3 1.:;5 * O.lGd
D Expressed as Karrnen units. One Karmen unit corresponds to a change of 0.001 units of absorbance at 340 urn in 3 ml of assay solution in a l-cm light path. * The activation coefficient is the ratio of enzyme activity in t,he presence aud absence of added pyridoxal-5’-phosphate (5 X 10e5 14f) incubated with the serum samples for 20 nun at 25°C before assaying. c Result,s are expressed as the mean of six chicks f SEX d 8ignifieantly difl’erent from contn& (P < 0.01).
RESULTS In the ad libitum feeding experiments, the pyridoxine-deficient chicks grew at a much slower rate than the controls. The control chicks in Expt I showed a mean gain of 55.2 g after 11 days while the deficient chicks had only increased in weight by 26.0 g. In Expt II, the controls at Day 8 had gained 32.7 g, whereas the deficient chicks had gained ouly 14.7 g and were no louger growing. The growth failure with pyridoxiue deficiency was related primarily to a marked reduction in food intake. In the restricted-feeding experiment the deficient chicks at 8 days had increased in weight by only 8 g and the control group was limited to a mean weight gain of 10.7 g. Six chicks from each group were killed at that point and the remainder continued on the same diet. After an additional 4 days (Day 12)) the remaining control and deficient chicks showed mean total gains of 17.5 and 18.3 g, respectively. Chicks on the deficient diet had neurological signs of pyridoxine deficiency after 7 or 8 days and these became severe by 10 or 11 days. Pyridoxine deficiency was confirmed by measurement of serum GOT activity (Table II ). Th e activities were substantially lower in deficient chicks in both ad Z&turn and restricted-feeding experiments, while the activation coefficients were significantly increased. The lysyl oxidase measurements for the ad Zibitum feeding experiments are summarized in Table III. In the first experiment a 75% reduction in enzyme activity was found in the phosphate buffer extracts of epiphyseal cartilage from deficient chicks after 11 days on the diet. In the second experiment chicks were raised for only 8 days before lysyl oxidase activity was measured in urea extracts of epiphyseal cartilage and aorta. Here a 57% reduction was found in the cartilage extracts but there was no apparent change in lysyl oxidasc activity in cstracts of aorta. To oliminatc any possible effect of the marked suppression of growth on lysyl oxidase in pyridoxine deficiency, a third experiment was carried out with similar growth suppressiou induced in the control chicks (Table IV). After 8 days, the total lysyl osidase activity of
PYRIDOXINE
DEFICIENCY
AND TABLE
Lyxyl
Oxidase
Activities
in Pyridoxine-Deficient Chick
weight
CHICK
LYSYL
305
OXIDASE
III and Cont.rol
Chicks
Lysyl
oxidase
Fed ad Lib&m activity”
(9) PBS extractb Expt I (11 days) Cartilage : control Pyridoxine-deficient Expt II (8 days) Cartilage : control Pyridoxine-deficient Aorta: control Pyridoxine-deficient
Urea
extractb
-
(6)c (6)
93.2 f 64.0 f
5.5d 5.0
11.8 3.5
(6) (6)
70.1 f 52.9 i
3.2 3.5
-
62.4 26.7
(6)
70.1 f 52.9 f
3.2 3.8
-
135.0 139.0
(6)
o Lysyl oxidase activity is expressed as unit,s per gram of wet weight of tissue, 1 unit (U) corresponding to the release of 100 cpm of tritium/8hr incubation at 37°C from 100,000 cpm of L[4,5-SHJysine-labeled aortic protein substrate. b Pooled samples of tissue were extract,ed with phosphate-buffered saline followed by 4 M urea0.05 M Tris-HCl (pH 7.5) at 25°C. In Expt I the salt extract was assayed while in Expt II only the urea extract was used. All assays were done in duplicate. c The figure in brackets is the number of chicks from which tissue was pooled for measuring enzyme activity. l1 Mean f SEM.
the epiphyseal cartilage of pyridoxine-deficient chicks was reduced by 49% compared to the value for growth-restricted controls. A greater drop was found in the activity in the salt-soluble extracts when compared with the urea-soluble extracts. There was no significant difference in enzyme activity of aorta extracts between the diet-restricted controls and the pyridoxine-deficient chicks, a result similar to that found at 8 days in the ad libitum feeding experiment. At 12 days the total activity of cartilage extracts of the controls had not changed, whereas the activity of this extract from the deficient chicks was now reduced by 60%. In addition, the lysyl oxidase activity of the aorta of pyridoxine-deficient chicks at Day 12 was 29% below that of the aorta of controls. In the deficient chicks repleted with pyridoxine 14 hr before sampling, the total 1ysyI oxidase activity of cartilage extracts was only 11% below that of the controls in contrast to the reduction of 60% in the pyridoxine-deficient chicks. Aorta extracts from the repleted chicks had 207 0 more activity than the controls. Attempts to reactivate lysyl oxidase in vitro by the addition of pyridoxalS’phosphate to cartilage and aorta extracts from pyridoxine-deficient chicks were unsuccessful. The distal epiphyseal cartilage of the tibiotarsal bone and proximal epiphyseal cartilage of the tarsometatarsal bone of the pyridoxine-deficient chicks appeared narrower than those of the control chicks; the columnar cartilage seemed disorganized and the lacunae were less uniformly arranged and more variable in size, confirming the description given by Gries and Scott (1972); the periosteal elastin appeared normal. The aortas of the pyridoxine-deficient chicks showed a generalized loosening of the texture of the laminar structure.
308
MURRAY,
FRASER,
AND
LEVENE
The spleen of the pyridoxine-deficient chicks showed a narrowing of the capsule which consisted mainly of collagen and elastin; it also appeared fragmented. All other tissues examined appeared normal. DISCLJSSION Lysyl oxidase activity in epiphyseal cartilage and aorta of the chick is diminished by pyridoxine deficiency and this effect cannot be explained by the suppression of growth of the deficient animals. The loss of activity was more pronounced in cartilage than in aorta which may reflect the relative predominance of collagen and elastin, respectively, as substrates in these two tissues. Lysyl oxidase is an extracellular enzyme (Layman et al., 1972) and binds tightly to an insoluble elastin matrix, thus itself becoming “insoluble (Kagan et al., 1974).” This enzyme-substrate complex may have the physiological benefit of protecting the enzyme from inactivation by immobilizing it. In the experiments reported here, the characteristic elastin affinity of lysyl oxidase was demonstrated by the observation that enzyme was only present in urea extracts of aorta, whereas a significant proportion of that in cartilage could be extracted with buffered saline. Because of the protective nature of the elastin-enzyme complex, it is possible that lysyl osidase in aorta may have a longer half-life than that in cartilage. TABLI.: Lpsyl
Oxidase
Artivities
in Pyridoxine-Deficient Chick
IV and Diet-Restricted
weight M
Lvsvl ., . PBS extract,b
Expt III At 8 days:
control
(6)c
s0.s
zt 1.6”
extract”
Total
32.9 136.0
37.5 136.0
Cartilage aorta
1.4 -e
17.9 1x0.0
19.3 130.0
3.9
Cartilage Aorta
7.6 -
31.0 147.0
38.6 147.0
AZ f.1
Cartilage Aort.a
1.7 -
14.0 104.0
15.7 104.0
Cartilage Aurta
6.9 -
27.4 180.0
x4.3 180.0
At 12 days:
(6)
57.5 f
(6)
5s.3
Pyridoxine-deficient then repleted
Urea
activit,y”
4.6 -e
(ti)
Pyridoxine-deficient
oxidase
Chicks
Cartilage Aorta
Pyridoxine-deficient
control
Control
and (z)!
45.5
5 Lysyl oxidase is expressed as units per gram of wet weight of tissue, 1 U corresponding to the release of 100 cpm of t,ritium/6-hr incubation at 37°C from 100,000 cpm of L-[4,5-sH]lysine-labeled aortic protein subst,rat,e. a Pooled samples of tissue were extracted with phosphate-buffered saline (pH 7.7) at 4°C followed by 4 121 urea-O.05 ,lf Tris-KC1 (pH 7.5) at 35°C. Both extracts were assayed separately. c Number of chicks from which tissue was pooled. d Mean f SEM. c Activities were too low t,o measure. 1 Two pyridoxine-deficient chirks were dnsed orally with 1.50 pg of pyridoxine 14 hr before sacrifice.
PYRIDOXINE
DEFICIENCY
AND
CHICK
LYSYL
OXIDASE
307
Although the tightly bound fraction of lysyl oxidase in cartilage was diminished in pyridoxine deficiency, the salt-soluble pool never became completely depleted. This suggested that there may be some interchange between the soluble and insoluble pools of enzyme activity in this tissue. When pyridoxine-deficient chicks were repleted with a large dose of pyridoxine, lysyl oxidase activity in both cartilage and aorta was rapidly elevated. This effect is unlikely to be caused by tissue growth, but rather it indicates direct stimulation of lysyl oxidase either by enhanced de nooo synthesis or by activation of the apoenzyme. A similar phenomenon has been observed in copper-deficient chicks in which 20 hr after administration of copper aortic IysyI oxidase activity rose from 5% of control to near normal levels (Harris, 1976). It was suggested here that in the absence of cofactor, the enzyme is more rapidly degraded, Enhancement of the enzyme activity when the cofactor is supplied in viva may result from decreased enzyme degradation together with continued and possibly increased enzyme synthesis. Since pyridoxine-dependent enzymes undergo rapid proteolysis in the absence of pyridoxal-phosphate ( Kominami and Katunuma, 1976), the explanation for the effect of copper may apply also to the enhanced lysyl oxidase activity found on repletion with pyridoxine. Degradation of most of the apoenzyme in the absence of its pyridoxal-phosphate cofactor would also explain the inability to reactivate in vitro the lysyl oxidase in tissue extracts. These results therefore indicate that both copper and a derivative of pyridoxine are essential cofactors for the lysyl oxidase enzyme. The presence of a pyridoxine cofactor in lysyl oxidase could help explain some of the pathology of connective tissues. Rinehart and Greenberg (1949, 1956) demonstrated the presence of atherosclerotic lesions in monkeys maintained on “suboptimal levels” of pyridoxine. Levene and Murray (1977) have recently suggested that in the developing human fetus, where pyridoxine IeveIs may be “suboptimal,” reduced lysyl oxidase activity might explain the presence of focal lesions in the intima of human coronary arteries, which are characterized by fraying and splitting of the internal elastic lamina (Levene, 1956). Copper-deficient chicks die suddenly from rupture of the aorta and other large arteries and severe morphological damage may be seen in these vessels from an early stage of ‘deficiency. Such severe changes are not found in pyridoxine deficiency for, unlike the copper-deficient animals, these show a sudden cessation of growth, caused mainly by inanition (Gries and Scott, 1972). Hence, most connective tissue protein was laid down before the onset of deficiency and crosslinking defects are not as apparent morphologically as in copper deficiency. ACKNOWLEDGMENT The authors are greatljr indebted and in particular to Mr. John Allen
to the Histology Section of the Department for his considerable technical expertise.
of Pathology
REFERENCES BAIJXY, A. J., ROBINS, S. P., and BALIAN, G. (1974). Biological significance of the intermolecular crosslinks of collagen. Nature (London) 251, 105-109. BLASCHKO, H., and BUFFONI, F. 1965). Pyridoxal phosphate as a constituent of the histamine (benzylamine oxidase) of pig plasma. Proc. Roy. Sot. Ser. B. 163, 45-60. BLASCHKO, H., BUFFONI, F., WEISSMANN, N., CARNES, W. H., and COULSON, W. F. (1965). The amine oxidase of pig plasma in copper deficiency. Biochem. J. 96, 4c. GALLOP, P. M., and PAZ, M. (1975). Posttranslational protein modifications, with special attention to collagen and elastin. Physiol. Reu. 55, 418487.
308
MURRAY,
FRASER,
AND LEVENE
GRIES, C. L., and SCOTT, M. L. ( 1972). Pathology of selenium deficiency in the chick. J. Nutr. 102, 1259-1268. HARRIS, E. D. (1976). Copper-induced activation of aortic lysyl oxidase in uioo. Proc. Nat. Acad. Sci. USA 73, 371-374. HARRIS, E. D., GONNERMAN, W. A., SAVAGE, J. E., and O’DELL, B. L. (1974). Connective tissue amine oxidase: II, Purification and partial characterization of lysyl oxidase from chick aorta. Biochim. Biophys. Acta 341, 332-344. HILL, C. H., and KIXI, S. C. (1967). The derangement of elastin synthesis in pyridoxine deficiency. Biochem. Biophys. Res. Commun. 27, 94-99. HUTTON, J. J., TAPPEL, A. L., and UDENFHIEND, S. ( 1966). A rapid assay for collagen proline hydroxylase. Anal. Biochem. 16, 384-394. KACAN, H. M., HEWITT, N. A., SALCEDO, L. L., and FI~ANZRLAU, C. (1974). A microenvironmental probe of elastin. Properties of a solubilized Congo red-clastin complex. Biochim. Biophys. Acta 365, 223-234. KARMEN, A. (1955). Transaminase activity in human blood. J. Clin. Inuest. 34, 126-133. KENNEDY, J. F. (1969). Liquid scintillation counting medium for aqueous samples. Erperientia 25, 1120. KOMINA~~I, E., and KATUNUAXA, N. ( 1976). Studies of new intracellular proteases in various organs of rats. Participation of proteases in degradation of ornithene aminotransferase in vitro and in uiuo. Eur. J. Biochem. 62, 425-430. LAYMAN, D. L., NARAYANAN, A. S., and MARTIN, G. R. (1972). The production of lysyl oxidase by human fibroblasts in culture. Arch. Biochem. Biophys. 149, 97-101. LEVENE, C. I. ( 1956). The early lesions of atheroma in the coronary arteries. J. Pathol. Bacterial. 72, 79-82. LEVENE, C. I., and MURRAY, J. C. (1977). The aetiological role of maternal vitamin-B0 deficiency in the development of atherosclerosis. Lancet 1, 628-629. MANNING, J. M., and MEISTER, A. ( 1966). Conversion of proline to collagen hydroxyproline. Biochemistry 5, 1154-1165. NARAYANAN, A. S., SIEGEL, R. C., and MARTIN, G. R. ( 1974). Stability and purification of lysyl oxidase. Arch. Biochem. Biophys. 162, 231-237. O’DELL, B. L., HA~DWICK, B. C., REYNOLDS, G., and SAVAGE, J. E. ( 1961). Connective tissue defect in chick resulting from copper deficiency. Proc. Sot. Exp. Biol. Med. 108, 402405. PAGE, R. C., and BENDITT, E. D. (1967). Molecular diseases of connective and vascular tissues: II, Amine oxidase inhibition by the lathyrogen, beta-aminopropionitrile. Biochemistry 6, 1142-1148. PINNELL, S. R., and MARTIN, G. R. (1968). The cross-linking of collagen and elastin: Enzymatic conversion of lysine in peptide linkage to alpha-aminoadipic-delta-semialdehyde (allysine) by an extract from bone. PTOC. Nut. Acad. Sci. USA 61, 708-716. RINEHART, J. F., and GREENBERG, L. D. ( 1949). Arteriosclerotic lesions in pyridoxine-deficient monkeys. Amer. 1. Pathol. 25, 481492. RINEHART, J. F., and GREER’BEHC, L. D. ( 1956). Vitamin BF deficiency in the rhesus monkey with particular reference to the occurrence of atherosclerosis, dental caries, and hepatic cirrhosis. Amer. J. Clin. Nutr. 4, 318-325. SHIELDS, G. S., COULSON, W. F., KI~~BALL, P. A., CARNES, W. H., CARTWRIGHT, G. E., and WINTROBE, M. M. (1962). Studies on copper metabolism: 32, Cardiovascular lesions in copper deficient swine. Amer. J. Pathol. 41, 603-617. SIEGEL, R. C., PAGE, R. C., and MARTIN, G. R. (1970). Th e relative activity of connective tissue lysyl oxidase and plasma amine oxidase on collagen and elastin substrates. Biochim. Biophys. Acta 222, 552-555. STANULOVIC, M., MILETIC, D., and STOCK, A. ( 1967). Die Diagnostik des Vitamin B,-Mangels auf Grund der Bestimmung von erythrocytsrer L-asparat: 2-Oxoglutarat Aminotranspherase ( Glutamat-Oxalacetet-Transaminase) und ihrer Stimulation ill vitro mit Pyridoxal5’-Phosphat. Clin. Chim. Acta 17, 335-362. STARCHER, B. C. (1969). The effect of pyridoxine deficiency on aortic elastin biosynthesis. Proc. Sot. Rxp. Biol. Med. 132, 379-382. TANZER, M. L. (196G). In “Biochemistry of Collagen” (G. N. Ramachandran, and A. H. Reddi, eds.), pp. l-37-162. Plenum, New York. YASVNOBU, K. T., and YAMADA, H. ( 1963 ). In “Symposium on the Chemical and Biologicar Aspects of Pyridoxal Catalysis” (E. E. Snell, ea.), pp. 453465. Pergamon, New York,
The
AND
Effect
hlOLECULAR
of Pyridoxine
J. C. *Department and fDunn
PATHOLOGY
of Pathology, University Nutritional Laboratory, Milton July
( 1978)
Deficiency on Lysyl Oxidase in the Chick
Activity
D. R. FRASER,~ AND C. I. LEVENE”~~
MuRRA%,*J
Received
28, 301-308
of Cambridge, Tennis Court Road, Cambridge University of Cambridge and Medical Research Road, Cambridge, England
6, 1977,
and in revised
form
October
CB2 IQP Council,
17, 1977
The activity of lysyl oxidase, the enzyme responsible for the production of the precursor of the crosslinks of collagen and elastin, was measured in the aortas and epiphyseal cartilage of chicks raised on pyridoxine-deficient and control diets. Lysyl oxidase activity was diminished in both tissues in the deficient chicks, although the activity declined more slowly in the aorta. Deficient chicks given a large dose of pyridoxine 14 hr before sacrifice showed a marked increase in lysyl oxidase levels in both tissues. It is concluded that a pyridoxine derivative as well as copper are essential cofactors for lysyl oxidase. INTRODUCTION
LysyI oxidase is the enzyme responsibIe for the oxidation of specific lysine and hydroxylysine residues in collagen and elastin to form the aldehyde precursors of the crosslinks in these important structural proteins. (For reviews see Bailey et al., 1974; Gallop and Paz, 1975; Tanzer, 1976). Lysyl oxidase therefore has a crucial role in determining the structural integrity of connective tissues. Crosslink defects of elastin, resulting in aortic rupture, have been demonstrated in copper-deficient animals (O’Dell et al., 1961; Shields et al., 1962). In addition to these defects, a pronounced decrease in plasma amine oxidase activity was also found in copper-deficient swine (Blaschko et al., 1965). Page and Benditt (1967) suggested that lysyl oxidase might belong to that group of amine oxidases which are known to be copper dependent and probabIy also pyridoxal phosphate dependent (Yasunobu and Yamada, 1963; Blaschko and Buffoni, 1965). Hill and Kim (1967) found that, in addition to a reduction in the amine oxidase activity of aorta from pyridoxine-deficient chicks, there was a corresponding reduction in the conversion of lysine to the crosslinks desmosine and isodesmosine. These authors therefore suggested that lysyl oxidase contains a pyridoxine derivative and that pyridoxine deficiency has the same effect upon elastin biosynthesis as copper deficiency. Starcher (1969) confirmed a reduction in the synthesis of crosslinks in elastin of pyridoxine-deficient chicks but, unlike Hill and Kim (1967), could find no change in total elastin. Because these earlier studies did 1 J.C.M. was the recipient of an MRC Research Studentship while 2 C.I.L. is a member of the External Scientific Staff of the Medical
doing this work. Research Council.
301 0014-4800/78/0283-0301$02.00/O All
Copyright 0 1978 rights of reproduction
by Academic Press, Inc. in any form reserved.
302
MURRAY,
FRASER,
AND
TABLE
LEVENE
I
Amormt (g/kg of die(.)
Q Vit.amin mi.xt.ure (lug/kg of diet,) : riboflavin, 15; thinmine hydrochloride, 15; calcium pantothenate, 20; folic acid, 6 ; hiotin, 0.6 ; cyanocohalmk, 0.02 ; nicotinxmide, 50; vitamin A acetate, 120 ; DL-wtocopheryl acetate 500 ; menaphthone, 40. Vitamin D given orally, .5 pg on arrival. b Brigg’s salt mixture (A. B. Cos, Brighton, Sussex, UK) : (g/kg of diet) ; CaCOs, 12.3; CnHPO+ 19.5; NazHPOI, 11.1; KCI, 12.3; RlgHOa, 4.2; RInS04, 0.27; ferric citrate, 0.261; ZnC03, 0.045; CuSO,, 0.0225; KI03, 0.0015.
not directly estimate the activity of lysyl osidase, we have investigated in the chick the possible influence of pyridosine deficiency on this enzyme using the specific tritium release assay of Pinnell and Martin (1968) in view of the high substrate specificity of the enzyme ( Siegel et al., 1970); we also examined the effect of pyridoxine deficiency on comlective tissue morphology. MATERIALS One-day-old maIe chicks of Light Sussex/Rhode Island Red stock were obtained from the National Institute for Research in Dairying (Shinfield, Berkshire, UK). L-[4,5-3H]lysine monohydrochloride (sp act., 8.7 Ci/mmole) was purchased from the Radiochemical Centre ( Amersham, Buckinghamshire, UK). h4ETHODS Chicks were kept in heated brooder cages supplied with drinking water. Experimental groups were raised on a pyridosine-deficient diet (Table I) modified after Gries and Scott (1972) whiIe control groups were fed the same diet supplemented with pyridoxine hydrochloride (9 mg of pyridoxine/kg of diet). In the first two experiments, control and deficient groups were fed the diets ad Zibitum. In a third experiment the diet for the control group was restricted to give the same growth rate as the deficient chicks. In this latter experiment oral doses of 150 pg of pyridosine were given on Day 11 to two deficient chicks which were then killed 14 hr later. At the end of each experiment blood was collected by jugular venesection and the serum was stored at -20°C. Aortas and epiphyseal cartilage from the leg bones were removed and frozen prior to measurement of Iysyl oxidase activity. Serum filutamntc-oxalonrctate-tm~~sarninnse n,Psq. Serum glutamate-oxaloacetate-transaminase (GOT) activity was estimated by the method of Karmen ( 1955). Serum samples were incubated in the presence and absence of added pyridoxal-S-phosphate (5 x 1O-5 iJ4) at 25°C for 20 min before assaying. The
PYRIDOXINE
DEFICIENCY
AND CHICK
LYSYL
OXIDASE
303
ratio of the activities of the serum with and without added pyridoxal-S-phosphate is the activation coefficient, aGoT,which can be used as an index of pyridoxine status (Stanulovic et al., 1967). Preparation of substrate for lysyl ox&se assay. Insoluble substrate from chick embryo aorta IabeIed in vitro with L-[4,5-3H]Iysine was prepared essentially by the method of Pinnell and Martin ( 1968). Aortas from 36 chick embryos at 17 days of development were dissected and incubated at 37°C for 8 hr in 10 ml of Krebs-Ringer buffer (Manning and Meister, 1966) supplemented with [L-45 3H Jlysine monohydrochloride (25 &i/ml), p-aminopropionitrile fumarate (50 pg/ml), ascorbic acid (50 rg/ml), benzylpenicillin ( 100 U/ml) and streptomycin sulfate (100 U/ml). After incubation the medium was decanted, the aortas were washed twice with deionized water, then lyophilized, and stored at -20°C until required. To prepare the substrate for lysyl oxidase assays, labeled aortas were suspended in phosphate-buffered saline (PBS; 0.1 M NaH,P04-0.15 M NaCI, pH 7.7) and homogenized in a ground glass homogenizer. The resulting suspension was centrifuged at 17,OOOgfor 10 min at room temperature, resuspended in PBS, and boiled for 10 min to destroy endogenous lysyl oxidase activity. After centrifuging once more, the pellet was suspended in PBS to give approximately 100,000 cpm of tritium/0.5 ml. Extraction of lysyl oxidase. Although some lysyl oxidase activity can be extracted from chick tissues with PBS, the majority is rendered soIubIe onIy if concentrated urea is present in the buffer (Harris et al., 1974; Narayanan et al., 1974). In the experiments on epiphyseal cartilage and aorta, the salt- and the urea-soluble enzyme activities were assayed separately. Tissue from each group of chicks was pooIed and homogenized in ice-coId PBS (2 ml/g) with a SiIverson homogenizer and then centrifuged at 20,OOOgfor 20 min. The supernatant was retained and the process was repeated. The two supernatants were combined and used as a source of soluble lysyl oxidase. The residue was then twice extracted with 4 M urea-O.05 M Tris-HCI (pH 7.5) at room temperature in a similar manner. The supernatants were combined and used as a source of ureasoluble lysyl oxidase activity. Assay of tysy’l oxida.se activity. Before assaying, all urea extracts were dialyzed exhaustively against PBS at 4°C. Enzyme preparations together with 0.5 ml of labeled substrate were incubated in stoppered tubes with shaking at 37°C for 6 hr. Total volume for each assay was 1.5 ml. The reaction was terminated by adding 0.2 ml of 50% trichloroacetic acid and the tritiated water was obtained by vacuum distillation (Hutton et al., 1966). Radioactivity was measured using Kennedy’s scintillant ( 1969) with a 30% counting efficiency. The lysyl oxidase activity is given in units where 1 unit (U) corresponds to the release of 100 cpm of tritium/6-hr incubation/lOO,OOO cpm of substrate. Total enzyme activity is expressed as units per gram (wet weight) of tissue. All assays were done in duplicate. Preparation of sections for examination by light microscopy. A broad sample of organs and tissues collected from control and deficient chicks were placed immediately after dissection into 10% formal saline; particular attention was paid to tissues containing much elastin or collagen. Paraffin sections were prepared and stained with Harris’ hematoxylin and eosin and others with Weigert’s elastin stain, followed by iron hematoxylin counterstain and Curtis’ substitute for Van Gieson.
304
MURRAY,
FRASER,
AND
TABLE Serum
(;lutamate-Oxaloacetate-Tra~lsamillase Pyridoxine-ljeficient Chick
LEVENE
II and Cont.rol
weight
GOT
(GOT) Levels Chicks activity0
in S-I jay
ol-GOT*
k) Expt
II
Ad libitum cant rol Pyridoxine-deficient Expt III Rest.ricted diet. aIntro Pyridoxine-deficient
70.1 * 52.9 f
X;?’ 3.8
X0.6 * 40.4 f
5.5 3.7
I .04 f I .47 f
0.02 0.09”
.X.8 * 1.6 49.5 f 2.3
86.3 * 56.8 f
2.2 2.X
1.03 3z 0.0:3 1.:;5 * O.lGd
D Expressed as Karrnen units. One Karmen unit corresponds to a change of 0.001 units of absorbance at 340 urn in 3 ml of assay solution in a l-cm light path. * The activation coefficient is the ratio of enzyme activity in t,he presence aud absence of added pyridoxal-5’-phosphate (5 X 10e5 14f) incubated with the serum samples for 20 nun at 25°C before assaying. c Result,s are expressed as the mean of six chicks f SEX d 8ignifieantly difl’erent from contn& (P < 0.01).
RESULTS In the ad libitum feeding experiments, the pyridoxine-deficient chicks grew at a much slower rate than the controls. The control chicks in Expt I showed a mean gain of 55.2 g after 11 days while the deficient chicks had only increased in weight by 26.0 g. In Expt II, the controls at Day 8 had gained 32.7 g, whereas the deficient chicks had gained ouly 14.7 g and were no louger growing. The growth failure with pyridoxiue deficiency was related primarily to a marked reduction in food intake. In the restricted-feeding experiment the deficient chicks at 8 days had increased in weight by only 8 g and the control group was limited to a mean weight gain of 10.7 g. Six chicks from each group were killed at that point and the remainder continued on the same diet. After an additional 4 days (Day 12)) the remaining control and deficient chicks showed mean total gains of 17.5 and 18.3 g, respectively. Chicks on the deficient diet had neurological signs of pyridoxine deficiency after 7 or 8 days and these became severe by 10 or 11 days. Pyridoxine deficiency was confirmed by measurement of serum GOT activity (Table II ). Th e activities were substantially lower in deficient chicks in both ad Z&turn and restricted-feeding experiments, while the activation coefficients were significantly increased. The lysyl oxidase measurements for the ad Zibitum feeding experiments are summarized in Table III. In the first experiment a 75% reduction in enzyme activity was found in the phosphate buffer extracts of epiphyseal cartilage from deficient chicks after 11 days on the diet. In the second experiment chicks were raised for only 8 days before lysyl oxidase activity was measured in urea extracts of epiphyseal cartilage and aorta. Here a 57% reduction was found in the cartilage extracts but there was no apparent change in lysyl oxidasc activity in cstracts of aorta. To oliminatc any possible effect of the marked suppression of growth on lysyl oxidase in pyridoxine deficiency, a third experiment was carried out with similar growth suppressiou induced in the control chicks (Table IV). After 8 days, the total lysyl osidase activity of
PYRIDOXINE
DEFICIENCY
AND TABLE
Lyxyl
Oxidase
Activities
in Pyridoxine-Deficient Chick
weight
CHICK
LYSYL
305
OXIDASE
III and Cont.rol
Chicks
Lysyl
oxidase
Fed ad Lib&m activity”
(9) PBS extractb Expt I (11 days) Cartilage : control Pyridoxine-deficient Expt II (8 days) Cartilage : control Pyridoxine-deficient Aorta: control Pyridoxine-deficient
Urea
extractb
-
(6)c (6)
93.2 f 64.0 f
5.5d 5.0
11.8 3.5
(6) (6)
70.1 f 52.9 i
3.2 3.5
-
62.4 26.7
(6)
70.1 f 52.9 f
3.2 3.8
-
135.0 139.0
(6)
o Lysyl oxidase activity is expressed as unit,s per gram of wet weight of tissue, 1 unit (U) corresponding to the release of 100 cpm of tritium/8hr incubation at 37°C from 100,000 cpm of L[4,5-SHJysine-labeled aortic protein substrate. b Pooled samples of tissue were extract,ed with phosphate-buffered saline followed by 4 M urea0.05 M Tris-HCl (pH 7.5) at 25°C. In Expt I the salt extract was assayed while in Expt II only the urea extract was used. All assays were done in duplicate. c The figure in brackets is the number of chicks from which tissue was pooled for measuring enzyme activity. l1 Mean f SEM.
the epiphyseal cartilage of pyridoxine-deficient chicks was reduced by 49% compared to the value for growth-restricted controls. A greater drop was found in the activity in the salt-soluble extracts when compared with the urea-soluble extracts. There was no significant difference in enzyme activity of aorta extracts between the diet-restricted controls and the pyridoxine-deficient chicks, a result similar to that found at 8 days in the ad libitum feeding experiment. At 12 days the total activity of cartilage extracts of the controls had not changed, whereas the activity of this extract from the deficient chicks was now reduced by 60%. In addition, the lysyl oxidase activity of the aorta of pyridoxine-deficient chicks at Day 12 was 29% below that of the aorta of controls. In the deficient chicks repleted with pyridoxine 14 hr before sampling, the total 1ysyI oxidase activity of cartilage extracts was only 11% below that of the controls in contrast to the reduction of 60% in the pyridoxine-deficient chicks. Aorta extracts from the repleted chicks had 207 0 more activity than the controls. Attempts to reactivate lysyl oxidase in vitro by the addition of pyridoxalS’phosphate to cartilage and aorta extracts from pyridoxine-deficient chicks were unsuccessful. The distal epiphyseal cartilage of the tibiotarsal bone and proximal epiphyseal cartilage of the tarsometatarsal bone of the pyridoxine-deficient chicks appeared narrower than those of the control chicks; the columnar cartilage seemed disorganized and the lacunae were less uniformly arranged and more variable in size, confirming the description given by Gries and Scott (1972); the periosteal elastin appeared normal. The aortas of the pyridoxine-deficient chicks showed a generalized loosening of the texture of the laminar structure.
308
MURRAY,
FRASER,
AND
LEVENE
The spleen of the pyridoxine-deficient chicks showed a narrowing of the capsule which consisted mainly of collagen and elastin; it also appeared fragmented. All other tissues examined appeared normal. DISCLJSSION Lysyl oxidase activity in epiphyseal cartilage and aorta of the chick is diminished by pyridoxine deficiency and this effect cannot be explained by the suppression of growth of the deficient animals. The loss of activity was more pronounced in cartilage than in aorta which may reflect the relative predominance of collagen and elastin, respectively, as substrates in these two tissues. Lysyl oxidase is an extracellular enzyme (Layman et al., 1972) and binds tightly to an insoluble elastin matrix, thus itself becoming “insoluble (Kagan et al., 1974).” This enzyme-substrate complex may have the physiological benefit of protecting the enzyme from inactivation by immobilizing it. In the experiments reported here, the characteristic elastin affinity of lysyl oxidase was demonstrated by the observation that enzyme was only present in urea extracts of aorta, whereas a significant proportion of that in cartilage could be extracted with buffered saline. Because of the protective nature of the elastin-enzyme complex, it is possible that lysyl osidase in aorta may have a longer half-life than that in cartilage. TABLI.: Lpsyl
Oxidase
Artivities
in Pyridoxine-Deficient Chick
IV and Diet-Restricted
weight M
Lvsvl ., . PBS extract,b
Expt III At 8 days:
control
(6)c
s0.s
zt 1.6”
extract”
Total
32.9 136.0
37.5 136.0
Cartilage aorta
1.4 -e
17.9 1x0.0
19.3 130.0
3.9
Cartilage Aorta
7.6 -
31.0 147.0
38.6 147.0
AZ f.1
Cartilage Aort.a
1.7 -
14.0 104.0
15.7 104.0
Cartilage Aurta
6.9 -
27.4 180.0
x4.3 180.0
At 12 days:
(6)
57.5 f
(6)
5s.3
Pyridoxine-deficient then repleted
Urea
activit,y”
4.6 -e
(ti)
Pyridoxine-deficient
oxidase
Chicks
Cartilage Aorta
Pyridoxine-deficient
control
Control
and (z)!
45.5
5 Lysyl oxidase is expressed as units per gram of wet weight of tissue, 1 U corresponding to the release of 100 cpm of t,ritium/6-hr incubation at 37°C from 100,000 cpm of L-[4,5-sH]lysine-labeled aortic protein subst,rat,e. a Pooled samples of tissue were extracted with phosphate-buffered saline (pH 7.7) at 4°C followed by 4 121 urea-O.05 ,lf Tris-KC1 (pH 7.5) at 35°C. Both extracts were assayed separately. c Number of chicks from which tissue was pooled. d Mean f SEM. c Activities were too low t,o measure. 1 Two pyridoxine-deficient chirks were dnsed orally with 1.50 pg of pyridoxine 14 hr before sacrifice.
PYRIDOXINE
DEFICIENCY
AND
CHICK
LYSYL
OXIDASE
307
Although the tightly bound fraction of lysyl oxidase in cartilage was diminished in pyridoxine deficiency, the salt-soluble pool never became completely depleted. This suggested that there may be some interchange between the soluble and insoluble pools of enzyme activity in this tissue. When pyridoxine-deficient chicks were repleted with a large dose of pyridoxine, lysyl oxidase activity in both cartilage and aorta was rapidly elevated. This effect is unlikely to be caused by tissue growth, but rather it indicates direct stimulation of lysyl oxidase either by enhanced de nooo synthesis or by activation of the apoenzyme. A similar phenomenon has been observed in copper-deficient chicks in which 20 hr after administration of copper aortic IysyI oxidase activity rose from 5% of control to near normal levels (Harris, 1976). It was suggested here that in the absence of cofactor, the enzyme is more rapidly degraded, Enhancement of the enzyme activity when the cofactor is supplied in viva may result from decreased enzyme degradation together with continued and possibly increased enzyme synthesis. Since pyridoxine-dependent enzymes undergo rapid proteolysis in the absence of pyridoxal-phosphate ( Kominami and Katunuma, 1976), the explanation for the effect of copper may apply also to the enhanced lysyl oxidase activity found on repletion with pyridoxine. Degradation of most of the apoenzyme in the absence of its pyridoxal-phosphate cofactor would also explain the inability to reactivate in vitro the lysyl oxidase in tissue extracts. These results therefore indicate that both copper and a derivative of pyridoxine are essential cofactors for the lysyl oxidase enzyme. The presence of a pyridoxine cofactor in lysyl oxidase could help explain some of the pathology of connective tissues. Rinehart and Greenberg (1949, 1956) demonstrated the presence of atherosclerotic lesions in monkeys maintained on “suboptimal levels” of pyridoxine. Levene and Murray (1977) have recently suggested that in the developing human fetus, where pyridoxine IeveIs may be “suboptimal,” reduced lysyl oxidase activity might explain the presence of focal lesions in the intima of human coronary arteries, which are characterized by fraying and splitting of the internal elastic lamina (Levene, 1956). Copper-deficient chicks die suddenly from rupture of the aorta and other large arteries and severe morphological damage may be seen in these vessels from an early stage of ‘deficiency. Such severe changes are not found in pyridoxine deficiency for, unlike the copper-deficient animals, these show a sudden cessation of growth, caused mainly by inanition (Gries and Scott, 1972). Hence, most connective tissue protein was laid down before the onset of deficiency and crosslinking defects are not as apparent morphologically as in copper deficiency. ACKNOWLEDGMENT The authors are greatljr indebted and in particular to Mr. John Allen
to the Histology Section of the Department for his considerable technical expertise.
of Pathology
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308
MURRAY,
FRASER,
AND LEVENE
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