Blood Vessels 12: 302-306 (1975)

Isoenzymes of Lactate Dehydrogenase in Varicose Veins O. M rhovä and I. P rerovsky Centre for Research on Cardiovascular Diseases (Director: Prof. L. H ejhal), and Institute of Clinical and Experimental Medicine (Director: Prof. P. MAlek ), Prague

Key Words. Veins • Varices • Lactate dehydrogenase fractions Abstract. The activity of lactate dehydrogenase and fractions of this enzyme has been estimated in human large saphenous veins. Typical varicosities were com­ pared in composition with normal veins from the same patients. The total LDH activity was significantly decreased in typical varicosities. Varicose veins further differed from macroscopically normal veins by a lower content of aerobic fractions of LDH and a higher content of anaerobic fractions. These results suggest that there is a disturbance in metabolism of the smooth muscle of the venous wall in varicosities which would make the diseased wall more dependent on anaerobic glycolysis than normal tissue.

In recent years, new data have been collected on the biochemistry of the venous wall of normal and varicose veins. Most attention has been paid to mucopolysaccharides and collagen. Svejcar et al. [1963] reported that varicosities contained less collagen and more mucopolysaccharides than normal veins. Later, it was demonstrated both biochemically and histochemically that enzymes involved in mucopolysaccharide metabolism, such as /¿-glucuronidase, //-/V-acetylglucosaminidase and aryl-sulphatase, are increased in varicose veins [N iebes and L aszt, 1971; U rbanova and PRerovsky , 1972]. More recently, N iebes [1972] has hown an increased level of these enzymes in the blood of patients with primary varices. Less attention has been paid to studies of the metabolism of the smooth muscle of the venous wall. Histochemical studies have shown that in

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Received: January 24, 1975; accepted: June 2, 1975.

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varicose veins there is a decreased content of enzymes of the Krebs cycle [U rbanova and PRerovsky, 1972]. Since the metabolism of smooth muscle in veins, just as that in arteries, is mainly dependent upon anaerobic glycolysis [L aszt, 1971] we have attempted to measure the activity of lactate dehydrogenase (LDH) and the separate fractions of this enzyme in varicose veins.

Methods The large saphenous vein was removed by surgery in 15 patients with primary varices (14 women and 1 man). Varicose parts of the veins were compared with macroscopically normal segments of veins taken from each of these subjects. The removed veins were rapidly cleaned of perivenous tissue and adventitia (under histological control), washed with ice-cold physiological saline and after drying on filter paper the vein was weighed. For determination of LDH activity, we used an Ultra-Thurax homogeniser to prepare 2°/o homogenate in physiological saline at pH 7.5. Fractionation of LDH required a preparation of a 20°/o homogenate in veronal-barbiturate buffer at pH 8.6. After 1 h extractions at 0-3 °C, the homogenate was centrifuged at 1,000 g and the supernatant was immediately used for further analysis. LDH activity was determined by the tetrazolium technique using neotetrazolium chloride and phenazine methosulphatc [M rhovA and Z ev. plenyi, 1965], The results were calculated on the basis of the protein content of the extract estimated by the method of L owry et al. [1951]. LDH separation was carried out by paper electrophoresis on W3 at a voltage drop of 6 V/cm for 4 h. The separated fractions were determined by the tetrazolium technique and the use of nitroblue monotetrazolium (NBMT) chloride and phenazin methosulphatc (PMS) and then eluted in acetone. Optical density was read on an Optica Milano spectrophotometer at 490 nm. These values of the LDH fractions were used to calculate the percentage distribution of separate fractions in the total enzyme sample [M rhov A, 1971]. The results were statistically evaluated using Student’s paired t-test.

Total LDH activity in varicose veins was markedly lower than that found in macroscopically normal venous segments, the decrease being on the average 20 ± 8.2% (p < 0.01). Separation of the LDH fractions showed that typical varices differed from macroscopically normal segments by a lower percentage content of aerobic fractions «1+, ( p < 0 .0 1 ) and a;J (p < 0.025) with a higher percentage content of anaerobic fraction as (p < 0.01) (fig. 1).

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Results

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•/.

a H2

a3

aA

a5

Fig. 1. Separate LDH fractions in varicose and normal venous segments. □ = Macroscopically normal segment; ■ = typical varicosity. Results expressed in per­ cent, the sum of all LDH fractions was taken as 100 "/o (mean ± SD).

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/

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8

The illustrative zymogram in figure 2 shows the separate fractions of LDH in the large saphenous vein. Venous segments were divided into pieces, 3-5 cm long. Segments 1-6 were macroscopically normal, seg­ ments 7 and 8 showed typical varicosities. The zymogram shows clearly the increased content of anaerobic LDH fractions in the latter segments.

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Fig. 2. The LDH zymogram in the large saphenous vein. 1-6 = Macroscopically normal segments; 7, 8 = varicosity.

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It has been shown that varicose veins are characterized by disturbances in the metabolism of collagen and mucopolysaccharides. Besides, there are histological findings of irregular arrangement of muscle fibres in some parts of the varicosities which are characteristic for the initial stage of the disease. L aszt [1972] measured glucose uptake, respiratory quotient and the formation of lactic acid in varicose veins and reported that a smaller percentage of glucose was metabolized by oxydation than in healthy tissue, so that the ratio of lactate formation to oxygen uptake was significantly higher in varicose veins than in control material. This shifts of metabolism towards anaerobiosis corresponds to the findings of a de­ creased activity of Krebs cycle enzymes [U rbanova and PRerovsky , 1972], These results are in good agreement with the present finding of increased anaerobic fractions of LDH on typical varicosities. Synthesis of the anaerobic LDH fraction is stimulated by a low oxygen tension and its biological function is to maintain activity even with an excess of lactate [D awson et al., 1964], These findings, therefore, suggest that the varicose vein is more dependent upon anaerobic glycolysis than the nor­ mal vein. Decrease of the total activity of LDH in varicose segments is very probably due to the depression of the glycine metabolism in damaged tissue [U rbanova and PRerovsky , 1972], In the present experiments, each varicose sample was compared with the macroscopically normal vein of the same subject. Since it has been previously shown [Svejcar et al., 1964; N iebes and L aszt , 1971; U r ­ banova and PRerovsky , 1972] that the biochemical composition of nor­ mal veins in individuals with varicosities are different from that of veins of completely healthy subjects, it is probable that if we compared our data from typical varices with data from venous segments from com­ pletely healthy persons, even greater differences would appear than those reported here. It would seem from the present results that in primary varices there is a disturbance not only of the metabolism of collagen but also of the smooth muscle of the venous wall. From previous reports, it is not yet possible to say which of these disturbances is primary. The main structure which maintains the shape of the vein is collagen, which would suggest that the primary disturbance should be sought for in collagen metabolism. On the other hand, L aszt [1971] has argued that the primary disturbance is in carbohydrate metabolism. A shift to anaerobic metabolism results

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Discussion

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in instability of lysozymes and increases the rate of washout of enzymes from the latter, which results secondarily in changes in collagen structure. This basic question, however, remains both a subject of controversy and of future research. References D awson , D. M.; G oodfriend , T. L., and K aplan, N. O.: Lactic dehydrogenase:

functions of the two types. Science, N.Y. 143: 929-932 (1964). L aszt, L.: Zur Biochemie der Venenwand (Vena saphena magna)); in B üttner und L eu Die Venenwand, morphologische, biochemische und pathophysiologische

Aspekte, pp. 105-111 (Huber, Bern 1971). L owry , O. H.; R osenbrough , N. L; F aar, A. L., and R andall, R. L: Protein meas­

urement with the Folin phenol reagent. J. biol. Chem. 193: 265 (1951). M rhov X, O.: Sexual differences in the cathodic (anaerobic) ‘M’ fraction of lactic

dehydrogenase in the rat aorta. Cas. Lék. ¿es. 110: 62-64 (1971). M rhovX, O. and Z emplenyi, T.: The effect of sex differences and gonadectomy on

some aortic enzymes of rat. Q. exp. Physiol. 1: 289-299 (1965). N iebes , P.: Determination on enzyme and degradation products of glycosamino-

glycan metabolism in the serum of healthy and varicose subjects. Clin. chim. Acta 42: 399^408 (1972). N iebes , P. et L aszt, L.: Recherches sur l’activité des enzymes dans le métabolisme des mucopolysaccharides des veines saphènes humaines saines or variqueuses. Angiologica 8: 7-16 (1971). Svejcar, J.; PRerovsky, L; L inhart, J., and K ruml , J.: Content of collagen, elastin, and hexosamine in primary varicose veins. Clin. Sei. 24: 325-330 (1963). U rbanovX, D., and PRerovsky, L: Enzymes in the wall of normal and varicose veins. Histochemical study. Angiologica 9: 53-61 (1972).

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Dr. O. M rhov X, Centre for Research on Cardiovascular Diseases, Budfjovicka 800, Prague 4 (Czechoslovakia)

Isoenzymes of lactate dehydrogenase in varicose veins.

The activity of lactate dehydrogenase and fractions of this enzyme has been estimated in human large saphenous veins. Typical varicosities were compar...
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