UNIVERSIT3~ DEGLI STUDI DI FIRENZE

ISTITUTO DI PATOLOGIA SPECIALE MEDICA E METODOLOGIA CLINICA (B)* (Direttore: Prof. P. ARCANaELI)

ISTITUTO DI FARMACOLOGIA ** (Direttore: Prof. A. GIOTTI)

CLINICA ORTOPEDICA ° (Direttore: Prof. O. SCAGLIETTI)

AMINO-OXIDASE AND DERMAL CONNECTIVE TISSUE IN SCOLIOSIS

LuPo ANDREOTTI * ROBERTO TOCCAFONDI *

FRANCA BUFFONI ** ALBERTO GIOTTI **

PlETRO BARTOLOZZI o PAOLO ARCANGELI *

INTRODUCTION

In recent years our knowledge of intra- and intermolecular coliagen cross-links has made considerable strides 3.4, 39. I t is now generally accepted that the formation of these cross-links is caused by the oxidative deamination of specific lysine residues in the telopeptide region of the 0~ chains of the tropocollagen molecule 3, 4.

I t is thought that the enzymatic oxidation of the ~ amino group of lysine is cata- lyzed by an amino-oxidase found in the plasma of mammals 19, 3o

This hypothesis is supported by both direct and indirect experimental evidence as follows:

1) it has been found that soluble amino-oxidases are inhibited by lathyrogens 6. 29;

2) it has been found that the amino-oxidases of blood plasma are copper-contain- ing enzymes, and it has been observed that copper deficiency is associated with al- terations in the structural stability of the connective tissue scleroproteins, certainly in laboratory animals ~, and probably in man as well (kinky hair syndrome) m. 27;

3) the collagen of various porcine tissues has been found to contain a protein with the same immunological properties as the amino-oxidase of plasma (benzylamino- oxidase) 5, 33;

4) it has been found that certain amino-oxidases are capable of increasing the in- tramoIecular cross-links 3. 8, 33.

5) a soluble amino-oxidase has been shown to exist in the aorta of cattle as, pigs 7, and dental pulp 22; it has been successfully isolated.

We have also shown that a non-mitochondrial amino-oxidase is present in the human skin, confirming the findings of LOVENBERG et at. 22, and some of its prop- erties are described.

Key-words: Amino-oxidase; Collagen cross-linking; Connective tissue; Scoliosis; Skin. Received, April 15, 1976. La Ricerca Clin. Lab. 6, 342, 1976.

342

L. ANDREOTTI et al.

40

"8

30

20

10 ¸

E

D

C o

B

A

0.033 0.066

benzylamine (mM)

Fig. 1 - Amino-oxidase activity of human skin. A, B, C, D, E and F refer to the cases.

We decided to study the degree of activity of this enzyme in human skin, both under normal and pathological conditions, and to correlate this oxidizing (amino- oxidase) activity with some biochemical properties of dermal connective tissue in idiopathic scoliosis.

We chose this disease as an example of a systemic disorder of the connective tissue, because it is a common clinical manifestation found in a wide range of heritable disorders of the connective tissue occurring, as it does, in many systemic mesenchymal disorders, such as Marfan's syndrome, the Ehlers-Danlos syndrome, osteogenesis imper/ecta 24, dystrophic nanism of LAMY and MAROTEAUX 20, and PINNELL's et aI. disease ~

Scoliosis also occurs in subjects apparently free from other disorders. Although we know so little about the pathogenesis of idiopathic scoliosis, it is highly probable that it should be included in the heritable disorders of connective tissue; indeed, this is implicit in the classification of some authors 28 who place scolioses that are un- doubtedly mesenchymopathic, such as Marfan's syndrome, among the idiopathic scolioses.

The principal argument to support this theory is the fact that a typical dorsal scoliosis can be produced in rats by treatment with amino-nitriles, the toxic compo- nents of Lathyrus odoratus seeds 27 which, as is well known, interfere with the collagen cross-linking mechanism. All attempts, however, to produce a scoliosis in laboratory animals similar to idiopathic scoliosis, either by muscular section or by a unilateral inhibition of growth, have been unsuccessful 17

Lathyritic scoliosis reproduces many essential features of idiopathic scoliosis, s u c h as :

1) a lateral deviation of the vertebral column;

2) rotation and wedge deformation of the vertebrae;

3) selective localization in the dorsal tract;

343

AMINO-OXIDASE IN SCOLIOSIS

4) and, above all, the fact that this symptom appears only during the growth phase 13

Pre l iminary histological observations z have shown a peculiar thinness of the colla- gen bundles in the skin of many patients wi th idiopathic scoliosis. This is in agree- men t wi th the above cited hypothesis of a degenerative pathogenesis in idiopathic scoliosis. This f inding led us to think that we might f ind diffuse alterations of the connective tissue in these patients.

MATERIALS AND METHODS

The following groups were studied: 1) 11 patients, aged from 28 to 49, suffering from various diseases other than scoliosis. This

group was used for a preliminary investigation of the amino-oxidase content of the skin; 2) 10 patients, aged from 12 to 18, with a severe idiopathic scoliosis; 3) 7 patients, aged from 13 to 25, with a severe poliomyelitic scoliosis. All the subjects in groups 2) and 3) were to undergo surgical correction of their scoliosis. A thin strip of skin, of a standard surface size measured in vivo, was removed from the edge of

the incision during the surgical procedure, and immediately cooled to almost 0 °C.; it was then minced with scissors and homogenized by an 'Uttra Turrax' homogenizer in 10 parts of phosphate buffer 0.06 M, pH 7.4.

Insoluble subcellular fractions were removed by centrifugation at 108,000 X g for 1 h. The amino-oxidase activity was measured by the MACCAMAN et al. method 24, the substrates

employed being both x4C-benzylamine 5.6 mCi/mmole and 14C-tyramine 20-40 mCi/mmole at various concentrations.

Radioactivity was measured in 15 ml of Bray solution in a Packard Tricarb spectrometer with the usual corrections for quenching. Over a period of 30 rain the enzymatic activity remained on a linear path.

Proteins were measured by the method of LowRY et al. ~ against a standard of BDH crystalline bovine serum albumin.

To calculate the kinetic parameters, Vm (maximal reaction velocity) and Km (apparent affznity constant), statistical methods were used x2, 40. Each measurement was made in triplicate with 4 different concentrations of benzylamine and tyramine. The standard error of this method is lower than 0.5 ~ .

To evaluate the collagen and elastin content of the skin we proceeded as follows: a portion of the strip of skin measured in vivo and of a standard size surface was weighed and homogenized by an 'Ultra Turrax' homogenizer. Skin collagen was fractionated by the slightly modified method of HoucK and JAcoB ~6. With this method it is possible to separate the following 4 fractions of dermal collagen:

1) neutral salt soluble collagen; 2) acid soluble collagen; 3) insoluble collagen; 4) wholly insoluble residue, composed of elastin and traces of keratin. To evaluate the collagen content of these fractions, their hydroxyproline content was multiplied

by 7.3 3~. The elastin content in the wholly insoluble residue was evaluated by multiplying the nitrogen content by 5.85, assuming a nitrogen content in the elastin of 17.1% ". No further puri- fication of the dermal collagen fractions was attempted. In all 4 fractions the nitrogen content was measured in quadruplicate by nesslerization according to HENgY ~5, after digestion with sul- phuric or perchloric acid 3~. Hydroxyproline content was measured in quadruplicate according to CONTE et al. 9. Carbohydrates were measured in duplicate by the anthrone method 4t, while the sulphur content was measured by an adaptation of LETONOFF and REINHOLD'S colorimetric method :t.

We measured the nitrogen, hydroxyproline and hexose in each collagen fraction. In the wholly insoluble residue, we only measured the nitrogen as an index of the elastin content, and the sulphur as an indirect index of keratin contamination.

This method makes it possible to split dermal collagen into 3 fractions with an increasing degree of polymerization: the number of cross-links is low in the neutral salt soluble collagen, medium in the acid soluble collagen, and high in insoluble collagen. By this method we obtained indirect evidence of the density of cross-links in coJ_lagen, and of the subdivision of dermal polysaccharides in the 4 separated collagen fractions. We have given the values in relation to the unit surface of the skin since, as shown by SHUSTEg and BOTTOMS 37, this is the only method by which significant comparative values can be obtained.

344

L. ANDREOTTI e t al.

RESULTS

In tab. 1 we give the amino-oxidase values of several samples of human skin. Within the range of the substrate concentrations used (between 0.03 and 0.66 mmo- les), the enzymatic activity agrees with the Michaelis-Menten equation. Michaelis' apparent constant is lower for tyramine than for benzylamine (tab. 1).

Tab. 2 shows how great the variations are in enzymatic activity (Vm) in the dif- ferent samples of human skin. The values of the apparent affinity constant (Kin) are far less variable.

Tab. 3 shows that the non-mitochondrial amino-oxidase of human skin is inhi- bited by:

a) ~-aminopropionitrile Iathyrogen;

b) diethyl-dithio-carbamate (a substance which has a chelating action on copper);

c) hyproniazide.

Tab. 4 gives the amino-oxidase activity found in the skin of patients with idio- pathic scoliosis and of patients with poliomyelitic scoliosis. Between these 2 classes of patients the differences found are statistically significant with a P ~< 0.04 when the analysis is accomplished according to SNEDECOR and COCHRAM 38 for 2 indepen- dent samples with different variance (t').

The Michaelis-Menten constant was not affected to any significant degree in sco- liosis, nor did the BIT ratio differ significantly in the 2 groups of scoliotic patients. In both, the oxidation of tyramine was much less than that of benzylamine (Vm of tyra- mine in idiopathic scoliosis = 1 ± 0.22 and in poliomyelitic scoliosis = 1.4 _+ 0.63).

In tab. 5 we give the following information for the patients with idiopathic or poliomyelitic scoli0sis:

1) the total collagen content and the respective amounts of tropocollagen, acid soluble collagen and insoluble collagen;

2) the absolute elastin content, and also the % elastin content in relation to the total collagen;

3) the total hexose content, its distribution, and the ratio of hexose to collagen within the 3 fractions.

There was a significant increase in the % of neutral salt soluble collagen in subjects with idiopathic scoliosis. In these patients there was also a positive correlation be- tween the hexose content in the neutral salt soluble fraction and mono-amino-oxidase

"C-benzylamine "C-tyramine c a s e

1"1o.

Km Vm Km Vm

1 0.35

2 0.35

3 0.29

4 0.15

31.6

50.8

35.2 8.2

0.15

0.05

0.07

0.22

5.30

7.90 0.40

2.20

Table 1 - Amino-oxidase in human skin. The Michaelis-Menten constant (K_m) compared with the maximal oxidization velocity (Vm) of benzylamine and tyramine. Km in mmoles; Vm in nmoles of oxidized substrate/mg of protein/0.5 h at 37 °C., pH 7.4.

345

CaSes s e x

A M

B M

C M

D M

E M

F F

G M

H F

I F

L M

M M

age (years)

49

61

64

32

51

60

48

56

40

56

28

AMINO-OXIDASE IN SCOLIOSIS

diagnosis

lipoma

abdominal hernia

abdominal tumour

lymphogranuloma

abdominal carcinoma

abdominal carcinoma

abdominal turnout

mammary carcinoma

mammary carcinoma

gastric ulcer

valvular heart disease

sample

dorsal skin

abdominal skin

abdominal skin

abdominal skin

abdominal skin

abdominal skin

abdominal skin

skin from the breast

skin from the breast

abdominal skin

gluteal skin

Km

0.14

0.15

0.35

0.14

0.19

0.37

0.13

0.21

0.40

0.08

0.14

Vm

43

54

53

447.6

1,086.0

1,160.0

224.5

132.0

1,030.0

36.6

21.7

Table 2 - Amino-oxidase activity in human skin. "C-benzylamine was used as the substrate; Km in mmoles; Vm in nmoles of oxidized substrate/g of wet tissue/0.5 h at 37 °C., plg 7.4.

activity (r = 0.748). In all cases the sulphur content of the wholly insoluble residue (and therefore the keratin contamination) was so low as to be negligible (ni trogen/ sulphur ratio in the insoluble residue = 0.254 i 0.06).

D I S C U S S I O N

Human skin contains an amino-oxidase which shows a marked similarity to the amino-oxidase of several connective tissues 7, 22.3< This enzyme is inhibited by copper- chelating substances, carbonyl reagents and ~-aminopropionitrile. This last inhibitor becomes active only at very high cop.centrations when tested against the crude skin extract.

The enzymatic activity of human skin catalyzes benzylamine oxidation at about 894 i 296 nmoles /g of wet t issue/h at 37 °C. These values agree wi th those of LOVENBERG et al. 22

W e cannot tell whether this enzymatic activity is due to one or several enzymes. Wi thou t doubt it is a soluble activity and not mitochondrial. W e cannot state posi- tively that the enzymatic activity observed is responsible for the oxidative deamina- tion of the amino a group of lysine in the tropocollagen molecule.

inhibitors

13-aminopropionit rile

diethyI-dithio-carbamate

hyproniazide

moles

10-'

I0-"

10-2

inhibition (%)

88 --* 3 (4)

18 + t (4)

98 ± 2 (4)

Table 3 - Amino-oxidase of human skin. l*C-benzylamine = 0.66 mmoles; average i standard error; the number of the experiments is given in brackets.

346

L. ANDREOTTI et al.

cases

A

B

no.

i0

5

benzylamine concentration (mmoles)

0.033 0.166 0.330 0.660

1.33 4.23 6.32 8.59 0.23 0.39 0.48 0.56

3.15 10.29 15.70 19.40 0.20 0.86 1.40 1.60

K m *

0.29

0.23

V m *

11.27

24.00

t' ** 2.18 2.61 2.70 2.70 (P = 0.05) 2.11 2.61 2.67 2.69

*Km and Vm were obtained with the method described by DOLARA and AGRESTI 12. Enzymatic activity: nmoles of oxidized substrate/mg of protein/0.5 h, average + standard error; ** according tO SNEDECOR a n d COCHRAM 38.

Table 4 - Amino-oxidase activity in the skin of patients with idiopathic scoliosis (A) and polio- myelitic scoliosis (B).

We found a decrease in the amino-oxidase activity in dermal extracts from patients with idiopathic scoliosis, in comparison with controls and the values given for normal human skin by LOVENBERG et al. 22

The positive correlation mentioned above between the hexose content of the neutral salt soluble fraction and the mono-amino-oxidase activity could indicate a closer correlation of amino-oxidase activity with the mucopolysaccharides of the ground substance, or with the unpolymerized neutral salt soluble collagen, rather than a correlation with insoluble collagen bundles.

Chemical separation of the dermal connective tissue of patients with idiopathic scoliosis allowed us to distinguish between 3 isolated collagen fractions which differ both physiologically and physicochemically.

The fraction of collagen soluble in neutral salts corresponds to tropocolIagen, i.e. to a monomeric collagen, entirely lacking, or almost entirely lacking, in stable inter- or intramolecular cross-links, t t has a rapid turnover, its biological half-life being approximately 24 hrs in the rat. Its concentration is high in young or growing tissues, decreasing gradually as the animal grows older, until in adult subjects it represents only a minimal fraction of the total collagen 36

Acid soluble collagen (sometimes called procollagen)36 is more polymerized; it contains many more cross-links than does tropocoIlagen, and the cross-links are es- sentially intramolecular. When there are any intermolecular cross-links, they are un- stable 4 and dissolve easily in an acid environment. This collagen is the main constit- uent of the tendons in the tails of rats. Its turnover is fairly rapid, but slower than that of tropocollagen (biological half-life in the rat = 5 days).

On the other hand, insoluble collagen has many stable inter- and intramolecular cross-links. Its tissue content and its polymerization would appear to increase deft- nitely during growth, and also in the adult phase of life. Its turnover is extremely slow (its half-life in the rat = 25 days).

From these characteristics it seemed reasonable to assume that the different pro- portions of the various collagen components in the tissues might provide informa- tion as to the greater or lesser number of cross-links in dermal collagen. Furthermore, collagen fibers owe their remarkable strength and their organization into bundles, not only to intermolecular cross-links, but also to the connexions which collagen

347

AMINO-OXIDASE IN SCOLIOSIS

protofibrils form, both with structural gtycoproteins, and with glucose-amino-glycans of the g round substance of connective tissues 26 with which, however, the connexions are weaker.

Accordingly we decided to study the hexose content of all 3 collagen fractions. I t is highly probable that the hexoses which can be extracted by 0.45 M NaC1 at a temperature of nearly 0 °C. belong to the glucose-amino-glycans of the ground sub- stance. I n hexose soluble in acid buffers, besides traces of glucose-amino-glycans left over f rom the first NaCI extraction, there are certainly some structural glyco- proteins bound to acid soluble collagen. The hexose which can only be extracted after hydrolysis of the insoluble collagen may presumably be considered to belong mainly to the structural glycoproteins which form closer connexions wi th insoluble collagen 2.

no . o~ cases

total collagen (mg/cm 2)

soluble collagen (% of the total)

soluble in neutral salts acid soluble

insoluble collagen (% of the total)

elastin (~g/mm z)

elastin collagen ratio (X 102)

idiopathic scoliosis poliomyelitic scoliosis

I0

33.3 ± 18.91

2.62 ± 124 *

1.79 4- 1.23

95.6 & 2.03

7.12 ± 4.89

2.69 ± 1.38

7

27.43 ± 6.28

1.60 ± 0.26 * 1.24 + 0.36

97.16 ± 0.49

7.51 ± 5.6

2.65 ± 1.76

* P < 0.001; the difference is highly significant.

hexose content in the skin of patients with

rlo. o~ cases

total hexose (~g/mm 2)

hexose extractable at low temperature (% of the total)

soluble in neutral salts acid soluble

hexose extractable from insoluble collagen (% of the total)

hexose content/lO0 mg of collagen soluble in neutral salts acid soluble insoluble

idiopathic scoliosis

9

25.41 ± 11.9

53.97 i 7.64 6.73 ± 2.31

43.404_10.22

24O.8 4-+- 104.27 62.38 + 44.49 3.76 ± O.73

potiomyelitic scoliosis

7

35.70 ± 11.5

34.68 ± 9.76 14.05 --+ 8.06

51.27 + 7.42

276.28 -+ 88.9 208.48 ± 183.75

7.49 + 3.93

Table 5 - Collagen, elastin and hexose content of the skin in patients with idiopathic and polio- myelific scoliosis. (Mean ± standard error).

348

L. ANDREOTTI et al.

If we analyze tab. 5, it becomes apparent that the o% of neutral salt soluble collagen is higher in idiopathic than in poliomyelitic scoliosis, and that the difference is highly significant (P < 0.00t). Although the mean age of patients with idiopathic scoliosis is somewhat lower than that of patients with poIiomyelitic scoliosis, the difference in neutral salt soluble collagen can only be partly attributed to a differ- ence in the age of the subjects, since the difference of age between the 2 groups is not statistically significant, and in the cases considered there was no significant corre- lation between age and the % of neutral salt soluble collagen in the skin. We found no statistically significant differences in the other 2 collagen fractions, nor in the elastin or hexose content in the 2 groups of patients.

The results obtained in the control patients with poliomyelitic scoliosis were on the whole homogeneous, whereas in the cases of idiopathic scoliosis there was a considerably greater variation in the total amount of collagen, and in the respective amounts found in the 3 isolated fractions. Significant differences in variability were also found in the amounts of acid-extractable hexoses, and for the ratio between hexose and both acid soluble and insoluble collagens.

From the findings reported the following conclusions may be drawn:

1) in idiopathic scoliosis, dermal collagen tends to be less polymerized than in the control group (poliomyetitic scoliosis);

2) the variability of the individual results suggests that idiopathic scoliosis is associated with a somewhat erratic mesenchymal maturation.

Accordingly, 2 working hypotheses can be put forward: one is that the label 'idio- pathic scoliosis' covers various different diseases which all show the same outward symptoms; the other that the organization of the connective tissue is disturbed in idiopathic scoliosis by some disorders in the collagen maturation process.

Further experimental work is needed to test these hypotheses. The statistically significant differences in the % composition of dermal collagen

in idiopathic scoliosis, in comparison with the controls, provides the first experi- mental evidence that idiopathic scoliosis may be a systemic disorder of the connective tissue.

SUMMARY

The amino-oxidase and collagen content of dermal tissue was evaluated in 10 patients with severe idiopathic scoliosis and in a control group of 7 patients with poliomyelitic scoliosis, that were to undergo surgical correction of their skeletal deformity. A greater dermal content of neutral salt soluble collagen was found in the patients with idiopathic scoliosis, compared with the con- trol group. Statistically the difference was highly significant. Mono-amino-oxidase activity, however, was found to be significantly decreased in dermal extracts obtained from patients affected by idio- pathic scoliosis in comparison with controls. The involvement of mono-amino-oxidase in the cross- linking of collagen and the possible classification of idiopathic scoliosis as a systemic disease of connective tissue are discussed.

ACKNOWLEDGEMENTS

We thank Mr. S. Sampognaro for technical assistance in connective tissue analysis. This work was supported by a grant from Florence University.

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AMINO-OXIDASE IN SCOLIOSIS

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PAOLO ARCANGELI Istituto di Patologia Speciale Medica

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Policlinico di Careggi Viale Morgagni 85, 50134 Firenze - Italia

351