Effect of vitamins K, D, and C on calcium and phosphorus in goat's milk. I. On the effect of vitamin K injection-香川大学学術情報リポジトリ

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Vol. 15, No. 2 (1964)

E F F E C T O F VITAMINS K,

D, AND C ON CALCIUM

AND PHOSPHORUS IN GOAT'S MILK

I On the Effect of Vitamin K Injection Toyoki MIYABE

Although a large number of reports have been published on the biochemical functions of vitamins K , D , and C to animals, the effect of these vitamins on the synthetic process of calcium phosphocaseinate in mamma1 y gland seems to remain almost unkown. The object of the present studies in this report is to elucidate the action of vitamins K , D , and C in relation to calcium metabolism.

In the experiment reported by YANAGISAWA('), it has been shown that the ion-calcium of blood serum permanently increased and the bound-calcium decreased when vitamin K was injected into the cervical vein of rabbit. A slight tendency of decrease of t h e calcium in the blood of cow, accordi~lg to OHARA('), is recognized, whereas t h e diffusible calcium exhi

-

bits the opposite tendency.

T h i s study described a s the first report presents data concerning the effect of vitamin K on the movement of the calcium and phosphorus to calcium phosphocaseinate i n milk when injected to the dairy goat.

Materials and Methods

Three Saanen dairy goats were used for this experiment which was made during the period from July t o November, 1961. Vitamin K s (menadione : 2-methyl- I , 4-naphthoquinone ) was the products of Takeda Yakuhin Co and its 120 or 200 mg was injected into the cervical vein of a female goat of Saanen breed belonging to the middle or later lactation period

.

Sampling of milk was done a t 8 or 24 hours during the lapse of time after injection early in the morning. Calcium was determined calorimetrically by Yanagisawa methodc3). The principle of this method is as follows : the color of reaction changes from violet to red when ion-calcium is added to a strongly alkaline solution of Plasmo Corinth B (disodium salt of 1 -hydroxy- 4 -chloro- 2, 2 -diazobenzene- 1, 8 -dihydroxy naphthalene- 3, 6 -disulfonic acid)

.

Phosphorus was determined by Fiske Subbarow methodc4). These emerging colors were measured in a T6kyG-k6den photometric calorimeter using 620 m,u (calcium) or 720 m,u (phosphorus) wave- length filter The diffusible calcium or phosphorus was expressed in the values obtained by the following method. First a glass pipe ( Icm in diameter by

5

cm in length) was prepared with fishskin membrane (Aidearu Co. ) a t its lower end. This membrane involved the 1 ml contents was tightly wrung by gum band. In this case, 0.5 ml of skimmilk was dialysed for 3 hours at 22OC through membrane against 3 L of distilled water, by stirring thrice a t regular intervals. The dialysed skimmilk was diluted accurately to 10 or 20 ml by adding the distilled water and analysed as samples

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1 50 Tech. Bull., F a c , Agr. Kagawa Univ.

for bound-calcium or bound-phosphorus. I n this procedure, since t h e sharp intake of dia- lysis percentage was shown at a point of 3 hours on the curved line, the values analysed at this point were expressed as calcium or phosphorus of diffusible form in milk. T h e nitrogen distribution was done by Rowland procedurec5). The milk protein was determined by Kjeldahl analysis for nitrogen, and the factor used for milk protein was 6.38 ( i . e . , 100/15.65).

Results

As shown in Table 1 , the intensity of alcoholic reaction of milks in both goats

I

and

II,

due to the injection of vitamin K within the limit of

120

to

200

mg, varied from the weak to the strong positive degrees. But there was no change in this reaction of goat

a.

With the laspe of time, on the other hand, the titratable acidity of milks showed a slight increase in all, not excepting the milk in goat

UI

.

Table 1. Effect of injection of vitamin K on titratable acidity and alcohol test of milk

No. Goat I

- .-

Amount of vitamin K 120 mg

I n Tables 2 to

4

are presented data showing the variation of protein or casein, ionization

of calcium or phosphorus, and ability of binding calcium or phosphorus to casein by injection of vitamin K .

Due t o the injection of 120 mg K to dairy goats, the phosphorus content exhibited an appreciable increase, although both the protein and calcium contents were not affected markedly or consistently with the lapse of time. But if observed in detail a s t o t h e protein content i n milk, this protein content was not entirely consistent, but i t decreased temporarily a t early

8

hours and then turned back to the initial state in the following 24 hours after injection. The casein content in milk, on the other hand, showed the same tendency to decrase as compared with protein content in milk.

A t

8

hours after injection, these reduced rates were both about 15 per cent for protein and casein contents respectively. Therefore, the distribution of casein ( i . e . , casein in 100 of total protein ) was not greatly altered a t

8

or

24

hours after injection.

As observed in goat I , it was recognized that the appreciable increase in phosphorus content occurred at the rate of about

20

per cent with little change in calcium content, at

8

or 24 hours inclusive. I n addition to the change of phospho~us content, the diffusible phoshorus content increased up to the maximum value (23 per c e n t ) , without affecting the diffusible calcium content Since the increases of phosphorus a n d diffusible phosphorus contents at

24

hours are relatively in parallel with each other, showing the rate of

20

and

Goat IT Goat

a

- A v e ~ a g e of

200 mg

1

200 mg

1 ,

goals l and

--

Hours after injection 0

___ __ I_ _

_

_-_ 8 24 0 8 -- - _ _- _ -

I

_15.0 _17.0 _{14 3} _17.0

+

4- L- i- Titratable acidity 70% alcohol test -.-- -15 5

+

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Vol. 15, No. 2 (1964) 151 23 per cent as the maximum values for each content, i t may be said that the increase of phosphorus content means that of diffusible phosphorus content.

Table 2. Effect of injection of vitamin K on protein, calcium, and phosphorus in milk

No.

1

Goat I

Amount of vitamin K 120 mg Hours after injection

I

0

1

8 Total protein g/dl 3.75 3.21 Casein g/dl 2 . 7 5 2.32 Casein i n 100 of total protein 7 3 . 3 72.3 Total Ca mg/dl 1125.0 1125 0 Diff. Ca mgjdl Diff

.

Ca i n 100 of total Ca Total P mg/dl 101.2 104.4 Diff. P mg/dl 54,7 46.9 Diff. P i n I O C i o f t o t a l P 54.1 44.9 Ca / P

/

1.24

1

1.20

As already described above, the low unit injection of vitamin K (120mg) is of little or Goat ]I

- . -

200 mg

of no importance insofar as calcium content is concerned, since its effect is so small as compared with phospho~us content. The values of Ca/P combined with casein were 1.10, 0.92, and 0.97 at 0, 8, and 24 hours respectively (Table 3 ) . The values of Ca/P were 1.24, 1.20, and 1.0.5 a t 0, 8, and 24 hours respectively (Table

2

)

.

A small and gradual intake of bound-Ca/P occurred with laspe of time. The value of the bound-Ca/P to casein was lower than that of Ca/P at the rate of about 0 4 7 per cent as average value, showing a definite trend toward a decrease in their ratio with the lapse of time. In this case, as described previously, the titratable acidity value showed a slight increase and the intensity of alcoholic reaction varied from the weak to the strong positive degrees.

Table 3. Effect of injection of vitamin K on calcium and phosphorus combined with casein. Goat

- -

200 mg

Average of goats IT and JlI

-- --

No.

1

Goat I

_-I__.-_ I

I-

--

Amount of vitamin K

1

120 mg 200 m g 200 mg

Due to the injection ( 120 mg

K)

, the calcium or phosphorus -combining capacity ( i. e., the bound-calcium or phosphorus in mg per I g of casein) increased temporarily a t 8 hours. Following this, they remained constant or rised slightly at 24 hours. At 8 hours, the

Average of goats 11 and Ill

Hours after injection

-

Bound-Ca(mg)percasein(g) Bound-P(mg)percasein(g) 8

1

24

1

0 8 24 0 8 24 24 - 18,8 19.3 - - - 12..3 16..3 0 15.0 14.0 Bound-Ca/P

1

1.10 -- - 0 18.5 16.9 0 9 7 0.92 8

-

22.9 24..8 - -- 16..1 ! 13.7 111.9 1.07 0.76

I

11.8 18.4 19..4 21.1 15.7 16.5 18..6 0.64 15.7 20.2 0.61 0.76 1 5 . 6 19.6 0,,74 0.97 0.76 0.79

-

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152 Tech. Bull. Fac Agr Kagawa Univ rable 4. T h e rates of increase and decrease after injection of vitamin K ,

No

..

1

Goat I

/

Goats 11 and

IU

Amount of vitamin K Hours a f t e ~ injection . . - -- - - - Total protein (%) Casein (%) 120 mg

I

200 mg -- -- - -- - 8

1

24

1

Ave

1

8

1

24 Ave - - -14 4 -0.3 -7 4 -12.5 -4 4 -8.2 -15 6

I

1 8

1

-6.9

1

-13.1

/

-4 5 -8 7 I Ave..

/

Ave. C a s e i n i n 1 0 0 o f totalprotein(%) -1.4

1

2.2

1

0.4

1

-1 3

1

-0.1

1

-0.7

1

-2 5

1

1 1 - -- -- - - - - - - -- - -. - - - -- - - Total Ca (%)

I

0 0

I

1 6

/

0 8

1

3 1

/

14.9

1

9.0

/

5 4

/

12.6 Dif f

.

Ca (%) Diff. Ca in 100 of total Ca (%) - - - -- -. Total P (%) Diff P (%) Diff P in 100 of total P (%)

casein content was not affected markedly by the same injection. From these facts, the action of binding the calcium to casein seems t o have a tendency to prevent the intake of casein production

.

AS the result of injection of 200 mg of vitamin K , the calcium, phosphorus, diffusible calcium, and diffusible phosphorus contents all exhibited apparently similar tendencies to increase as time passed by. Of these constituents, the phosphorus content showed a tendency to increase more evidently than the calcium content, even though both the calcium and phosphorus contents increased considerably. Pronounced increase in phosphorus content was rather found more a t 24 hours than a t

8

hours after iniection. T h e average rates of calcium increase in goats I1: and

IlI

were about 3 per cent a t

8

hours and about 15 per cent a t 24 hours, whereas those of the same phosphorus were about 9 per cent a t

8

hours and about 23 per cent a t 24 hours, respectively. Further tendency was more clearly recognized in goat

II

than in goat

lU.

The average rates of diffusible calcium increase in goats

I1:

and

lU

were about 26 per cent a t 8 hours and about 32 per cent a t 24 hours, whereas those of the same diffusible phosphorus were about 25 per cent a t 8 hours and about 30 per cent a t 24 hours. On the other hand, the tendencies exhibited t o decline the casein content in milk. This effect was considered to be caused by an increase in the diffusible calcium or phosphorus in milk, since it is not combinative to casein due to the presence of vitamin K in gland tissue.

These results, in other words, seem to suggest that vitamin K interferes with the s y n - thetic process of protein in relation to the producing ability of calcium phosphocaseinate in mammary gland, so that less casein was synthesized As for the casein distribution ( i - e , casein in 100 of total protein) had no particular variation regardless of the decrease of casein. The average rates of reduced percentage for the protein and casein contents were

8

per cent (max. 21 per cent) and

8

per cent (max. 22 per cent) , respectively. Thus i t is obvious that the decrease in protein is mostly due to the decrease of casein which

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may occur in gland tissue.

It was found that the remarkable ionization of calcium or phosphorus in milk did not affect the absolute amount of binding calcium or phosphorus t o casein, although the production of calcium phosphocaseinate decreased ; so that the bound-calcium or phosphorus in mg per

I

g of casein was not affected. Nevertheless, t h e phosphorus-combining power against casein was slightly promoted a s compared with that of calcium, as found in goat

a .

In other words, i t may be considered that the deficiency of production of calcium

phosphocaseinate by gland tissue was accompanied by the pronounced ionization of calcium or phosphorus.

Summary

The study reported here was initiated fox the purpose of investigating more thorough1 y the effect of vitamin K on the manner of calcium and phosphorus to calcium phosphocaseinate in milk when injected to the dairy goat.

Vitamin Ks (menadione) within the limit of 120 to 200 mg was injected into t h e cervical vein of female goat belonging to the middle or later lactation period.

As the results of iniedtion of 200 mg of vitamin K , the calcium, phosphorus, diffusible calcium, and diffusible phosphorus contents in milk all exhibited similar tendencies to increase as time passed by And the phosphorus content alone showed a tendency to increase more evidently than the calcium content, even though both the calcium and phosphorus contents increased.

On the other hand, the tendencies exhibited t o decline the casein content. This effect was considered to be caused by increase in the diffusible calcium or phosphorus in milk, since i t is not combinative to casein due to tbe presence of vitamin K in mammary gland. ~ u t the remarkable ionization of calcium or phosphorus in milk did not affect the amount of binding calcium or phosphorus to casein.

The low unit injection of vitamin K (120 mg) is of little or of no importance insofar as calcium content is concerned, since its effect is so small as compared with phosphorus content.

(Received December 10, 1963)

References

(1) YANAC;ISAWA, F., : J . J a p , Soc.. Food N u t r . . , TakyB, Bunkad8 (1953).

7, 37 (1954). (4) SUBBAROW, F.. S. : , J . Biol.Chem.. , 66.. 375

(2) OHARA, H.. , YOSHIDA, N . : Chikusan no (1925).

Kenk,ya, 10, 33 (q956). (5) ROWLAND, S. J. : J.Dai'r,y R e s . , 9 , 42 (1938).

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