From the Biochemical Institute of the University, Aarhus. Denmark.

The Cholesterol Content in Rabbit Seruni. BY

J0RGEN HOFFMEYER.

Received 4 March 1944.

The following paper deals with the cholesterol content in rabbit serum under normal and certain experimental conditions. The order of magnitude of the individual variaLtions and the daily variations in one and the same test animal are elucidated. A report is given of experiments showing the influence of different degrees of inanition, bleeding anaemia, ether narcosis and dif- ferent surgical operations OP the cholesterol content in serum. Such investigations are necessary for the evaluation of the altcr- ations which can be demonstrated in connection with adrenalec- tomy and hansplantation of adrenals, which will be the subject of a folIowing paper.

Material and Technique. In all experiments white rabbits have been used. The average weight

of the animals has been about 2 kg. The animals were kept in separate cages under constant temperature. The normal diet has been kohlrabi and hay. The animals were kept on the diet for more than a week before experimentation. The animals were weighed each morning before feeding. In the diet experiments the weight of food eaten by the animal was determined very accurately. The cholesterol analyses were carried out according to BRUN’S method (1935) in which both total and free cholesterol are determined. In this paper only the total cholesterol values are given. The haemoglobin concentration in blood was determined by the sicca-method (HESSE and TRIER 1937). The in- accuracy of both methods was less than 2 per cent.

314 JsRGEN IIIJFYMESER.

Experimental.

I. Indiv idua l Variations and the Daily Variations

Several authors state that the serum cholesterol varies con- siderably in different rabbits (SV. HANSEX 1927, GRIGAUT 1913, STARUF 1937, TEILUM 1940) whereas only a few authors (XORI and REISS 1928, STARUP 1937, TEILUM 1940) seem to have %at- ched the same rabbit over a longer period. It is not possible from the works quoted above to state the magnitude of individual variations and the daily variations in one and the same rabbit. and therefore the following investigations were undertaken.

Single decerminations of serux cholesterol were carried out on 211 white land rabbits. The average value W L ? ~ found to be 40.8 mg:/O with a standard deviation of 14.6. There seems to be a slight difference between male and female (male rabbit (in all 109): 35.3 i. 13.1 mgO/,, female rabbits (in all 102): 46.8 & 13.8 rug%). The difference between male and female is sipni- ficant; the standard deviation of the difference of the two means is 1.85.

Table I.

Daily Variations i n the Serum Choksterol Content in 1 2 Rabbits

in One and the Same Animal.

- lab- ' bit ~ Sex NO.

2,300 2,350 1,800 2,060 1,700 1,740 1,960 2,100 1,790 1,950 1,830 1,730

Total serum cholesterol in mg per cent, D&Y

1 I I ~I

27.81 31.9 31.91 32.0 36.8 32.i 35.i1 35.2 35.4 26 9 32 6 10 3

19.9 20.8 247 ' 24.5 27.9 31.1 23.1'30 8 25 5 27 9' 26.0 14 7

33 8 3S.9 26.6 26 0 28.9 24.6 30.7 36.1 26.6129.8 34.7 34.4

24.6 32.4 35.2' 32.6 28.4 27.9

21.1 31.1 24.OI29.0 19.9

47.9 41 3 45.6' 43 5 40 8 47.1 26.2 29.6 26 5 l 23.1 20.4

I 32 S I 32.7~ 44.7 55.2 52.4' 3.6 40 0 25 2

41.31 46 21 48.5 39.i 41.6 41.3, 41.4 9 61

41.3~ 43.8 48.5 43 5 37.2 41 i l 43.5 5.6 40.2146.8 4 7 4 46.2 51.21433 9 5

THE CHOLESTEROL CONTENT IN RABBIT SERUM. 315

The results of the investigations into the daily variations in the same animal are given in detail. 1 2 animals were watched for a period of 10 days with one double determination per day. 3 ml. blood were taken daily.

It appears from Table I that the serum cholesterol shows varia- tions from day to day in the normal rabbit. The magnitude of these variations is different froin animal t o animal and the standard deviation of the average value for the period of 10 days varies from 5.8 to 25.2 per cent. The single fractions of total cholesterol vary in the same degree as total cholesterol. The variations are of the same order of magnitude in male and female animals, and within the same litters, viz. 1, 2, 3, 4, 5 and 28, 29, 30, 31 belong to same litters.

11. The Influence of Bleeding Anaemia on the Cholesterol Content in Serum.

It is a well known fact that bleeding is followed by hypercholeste- rolemia (HORIUCHI 1920, BLOOR 1920, STARUP 1937). STARUP demonstrated that the hypercholesterolemia is due to oxygen deficiency, caused by the anaemia, It is of interest to find out the degree of anaemia which involves an increase in the serum cholesterol. Therefore the following experiments were carried out in which the animals were subjected to different degrees of bleeding dailj-.

The animals in Table I were bled 3 ml. daily without any increase in the serum cholesterol. It is se.en froni Table 11, how- ever, that daily loss of 6 to 15 ml. blood is followed by a hyper- cholesterolemia. Rabbits 72 and 74 thus show incipient hyper- cholesterolemia after 7 ml. blood have been drawn daily for 5 or 6 days. It should be mentioned that increase occurs when haemoglobin has fallen to 50 to 55 per cent and reaches maximum a t about 30 to 35 per cent. Continued bleeding does not involve a further decrease in haemoglobin, the rabbit now being able to compensate for the loss of blood. Rabbit No. 7 has a pronounced capacity for blood regeneration, a loss of blood of 15 ml. daily being necessary to obtain a haemoglobin content of 35 per cent. The three rabbits in Table I1 all show incipient hypercholestero- lemia a t 50 to 55 per cent haemoglobin, which may be considered the limit of risk of incipient hypercholesterolemia caused by anaemia. It should be mentioned that investigations on free

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THE CHOLESTEROL COBTENT Ih’ RABBIT SERUM. 31i

and esterified cholesterol showed an equal increase of both during these experiments.

2,300

111. The Influence of Different Degrees of Inanition on Serum Cholesterol.

ROTHSCHILD (1915), SHOPE (1927) a. 0. have demonstrated that absolute inanition will provoke a rapidly appearing hyper- cholesterolemia. These results are confirmed in Table 111, con- taining the data for three rabbits, which after a fore-period of two days do not get any food for 3 or 4 days. The increase in serum cholesterol amounts to 2 to 3 times the initial value.

2,370 2,025 1 1 1

Table 111.

T h e In f luence of absolute I n a n i t i o n o n Serum Cholesterol.

R a b b i t No. 8. Totalchol. mg % . . . . . . . Weight in g . . . . . . . . .

R a b b i t S o . 32. Totalchol. mg % . . . . . . . Weight in g . . . . . . . . . .

R a b b i t hTo. 3.7. Totalchol. mg % . . . . . . . Weight in 8. . . . . . . . . .

Before inanition During inanition

2 1

25.2 25.i 2,490

44.4 2,400

46.6

2,500

44.7

53.1 %,1% 2,190

1 / 8 / 3 4

Studies on partial inanition have not previously been reported. I n connection with surgical operations the appetite of the rab- bits and their intake of food may be considerably decreased. It was therefore natural to examine the influence of different degrees of inanition. The results are to be found in Table IV.

This table includes 2 animals, each of which in a fore-period have got a sufficient amount of food (400 g of kohlrabi). After the fore-period rabbit No. 7 got 100 g kohlrabi for 8 days (‘25 per cent of the normal intake) and No. 73 200 g kohlrabi for

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THE CHOLESTEROL CONTENT IN RABBIT SERUM. 319

9 days (50 per cent of the normal intake). In both cases a con- siderable hypercholesterolemia occurs, in rabbit No. 7 after 4 days and in rabbit No. 73 after 7 days. As the haemoglobin content of the blood in these animals does not decrease below 55 per cent the hyparcholesterolemia must be supposed to depend upon the reduced intake of food and not upon anaemia.

IV. The Influence of Ether Narcosis and Surgical Operations

Ether narcosis in man is followed by a hypercholesterolemia (MAHLER 1926, BLOOR 1914, REICHER 1908). ROTHSCHILD (1914) and LANDAU (1915) could not demonstrate a definite effect of ether narcosis on blood cholesterol in rabbits and BAUMANN and HOLLY (1923) found no change in rabbits after ether narcosis combined with different surgical operations. BAUMANN and HOLLY have only carried out a few examinations, but the author has been able to confirm their results.

In Table V are given the data for two rabbits (No. 7 and 12) which have been narcotized with ether for half an hour (35 ml. ether). No changes in serum cholesterol could be demonstrated during the days following the narcosis.

Table V also contains the results of experiments on four other rabbits. On rabbits No. 3 and No. 4 in ether narcosis 16 small pieces of psoas musculature were transplanted into the abdominal wall and on No. 9 and No. 10 a laparotomia with palpation of the intestines and adrenal regions were carried out. Neither of these procedures were followed by changes exceeding varia- tions in normal variations in rabbit serum cholesterol.

on Serum Cholesterol.

Discussion.

The experiments in this paper form the necessary basis of investigations on the influence of the adrenal cortex on the cholesterol content in rabbit’s serum. The serum cholesterol varies considerably in different rabbits and even in the same animal relatively big variations can be stated from day to day. These daily variations have not previously been reported. On a large material of white rabbits it is proved statistically, that the average value for serum cholesterol differs for the two sexes, the average value being bigger for female rabbits. This result

320 JMRGEN HOFFiMEYER.

I ~

I

2,505~ 8,460 2,4351 2,360' 2,410

95 92, 85' SO SO'

2,890 2,910 2,930 2,Y65 2,820 25.51 25.6 26.1 28.6 28.2

95, 931 901 80 80

54.SI 51.2' 47.G 50.7 49.8

I 1

2,200l

Table Y.

[ h e Influence of Ether Narcosis alone and combined with surgical Operations o n Serum Cholesterol.

,,

I

2,380' 2,385 2,330 2,350

781 75 75 75 49.11 48.9 51.2' 49.0

I

2,810 2,770 2,860 29.1 31.3 30 7

8 0 ~ 80 72

2,0%0

i I

R a b b i t A-0. 7. Weight in g. . . . . Totalehol. rng % . . Haemoglobin. % . . .

R a b b i t X o . 12. Weight i n g. . . . . Totalchol. mg % . . Haemoglobin. % . . .

R a b b i t hro. 3. Wright in g, , . . , Totalchol mg % . . .

R a b b i t XO. 4. Weight in g. . . . . Totalchol. mg 74 . . .

R a b b i t Xo. 9.

Weight in g. . . . . Totalchol. mg % . . .

R a b b i t S o . 10. Weight in g. . . . .

I Totalchol. mg % . . .

Before narcosis After narcosis

is a confirmation of the results of HANSEN (1927). Daily bleeding which is followed by a decrease in blood haemoglobin below 55 per cent is followed by hypercholesterolemia, while STARUP'R (1937) results seem to indicate that hypercholesterolemia does not start before the degree of 35 per cent haemoglobin. A nioderatc reduction of food intake, too, is followed by hypercholesterolemia. -

I n rabbits No. 3 aud No. 4 the ether narcosis v a s combined with trsns- plantation into the abdominal wall of 16 pieces of psoas musculature and in rabbits No. 9 and No. 10 with laparotoniia with palpation of intestines and adrenal regions.

THE CHOLESTEROL CONTEKT IS RABBIT SERUM. 321

Both moderate anaemia and pnrtlal Inanition arc thus soiirces of error which must be taken into consideration in work dealing with the part played by adrenal cortex on serum cholesterol. Neither ether narcosis alone nor ether narcosis combined with surgical opcrations correspouding to cstirpation and transplanta- tions of adrenals seem to influence the serum cholcsterol conccntra- tion.

Summary.

The cholesterol content has h e n determined in the serum of more than two hundred white land rabbits. The average value for all animals was found to be 40.8 nig per cent, the averagc value for the fernale rabbits being about 10 mg per cent higher than for male rabbitn. 11 report is given of espcriiiients showing varia- tions in the same animal froui day t o day. Further the iilfluence of different degrees of bleeding anaemia, inanition, ether mrcosis and surgical operations is described.

Beferences.

BAVMASN, E. J. and 0. R.1. HOLLY, J. Biol. Cheiii. 1933. 55. i57. BLOOK, W. D., Ibidem. 1914. I!). 13. BLOOR, W. D., Ibidem. 1920. 45. 171. BRUN, G . C., Hosp.tid. 1933, 68s. GRICAUT, h., Le cycle de la cholestbrinimie. Thise, Paris 1913. HANSES, Sv., Acta. chir. scand. 1927. 62. 483. HESSE, H. and M. TRIER, Ugeskrift f. I ~ c g e r 1037, 99. 935. HORIIXHT, Y., J. Biol. Cheni. 1920. 4sC. 373. LANDAU, M., Die Nehennierenrindc. Jciin 1915. MAHLKR, A. J., J. Uiol. Chciii. 1926. 69. 653. MOM, S. and M. REISS, Endokriuologir, 1928. 1. 418. HEICHER, K., Z. klin. RIed. 1908. 65. 835. ROTHSVRILI), RI. A., Beitr. path. Anat. 1914, ‘55. 667. ROTHSCHILD, RZ. A., Ibidem. 1915. 60. 227. SHOPE, R. E., J. Biol. Chem. 1927. 7;. 101. STARIT, U., Underriigelser over experimentel Hyperlipmiii. Disp.

TEILUM, G., Om hormorial Cholesteri~i:c~iii. Disp. Kolenhavn 1940. Kobenhaw 1937.