Vitamin D-ascorbIc Acid Association in Bone Metabolism 



49 



portion of bone. An increase in osteoclastic population (Levy and Gorlin, 1953) and 

 a decrease in bone alkaline phosphatase (Gould and Schwachman, 1941) have also 

 been shown in ascorbate deficiency. The present report concerns possible bone cellular 

 roles for vitamin D and ascorbic acid and an attempt to relate these events to bone 

 demineralization. 



Methods 



Male Leghorn chicks were given the experimental diets (Tab. 1) from one day 

 until 15 and 16 days of age. Previous efforts (Thornton et ai, 1959) attest to the 

 adequacy of the control diet to support normal growth and to its rachitogenicity 

 in the absence of vitamin D., . At 15 and 16 days of age, an equal number of chicks 

 from each group were sacrificed. The tibiae were quickly removed, freed of adhering 

 tissue and placed in cold saline solution. Each were split lengthwise and the bone 

 marrow removed. About 100 mg of compact bone tissue was placed in 2.8 ml of 

 cold buft'er solution and kept there until all samples were placed in the incubator. 

 The medium, adjusted to a pH of 7.4, contained the following in //moles: Tri- 

 (hydroxymethyl)aminomethane (Tris) 20, MgS04 15, glucose 10, NaCl 150, adeno- 

 sine diphosphate (ADP) 10 and hexokinase at 0.5 mg per flask. Tissue from 6 animals 

 from selected groups (Tab. 1) were incubated in the Warburg respirometer. All others 

 were incubated in the Dubnoff metabolic shaker with air used as the gas phase in 

 each case. All were incubated for 3 hours at 37 "C. 



Table 1. Dietary changes and bone response 



Dietary 



Treatment 



|j.g/100 mg Bone 



Percent 

 Bone Ash 



O2 uptake ' Glucose uptake 

 (xl/mg NCN/lu-. [xg/mg NCN/hr. 



Lactic Acid 



recovery 



ixg/mg NCN/hr. 



530 4:171 63.8^0.3 

 760±3la« 58.4 ±0.7' 



1. Control 



2. Vitamin D3 

 deficient .... 



3. 2 + 44 mg ascorbic 

 acid/kg diet. . . 849±41«" 58.5 + 0.5 "» — 



4. 2 + 220 mg ascorbic 



acid/kg diet . . . 564^44"'' 60.2 ± 0.4""''' 31 ± 5 



22 ± 51 859 ± 30 



51±10«« 451 ±49"' 

 — 470 ±29"' 



98 ±9 

 70±9« 

 70 ±8" 



712 ± 54&«''« 93 ± 10 



' Mean + S. E. 



a, aa — Different from the control at the 95 and 99''/o confidence levels respectively. 



b, bb — Different from the vitamin Dj deficient group at the 95 and 99% confidence levels respectively. 



Chemical determinations of the media included glucose (Huggett and Nixon, 

 1957), lactic acid (Barker and Summerson, 1941), phosphate (Fiske and Subbarow, 

 1925) and calcium (Willis, 1961). Non-collagenous nitrogen (NGN) was measured, 

 employing a modification of the Lilienthal et al. method (1950) as used by Borle 

 et al. (1960). 



Gompact bone tissue was ether extracted for 6 hours, dried to a constant weight 

 at 105 °C and ashed 12 — 16 hours at 600 G to determine percent ash. 



Results and discussion 



There was an apparent increase in NGN in the absence of vitamin Dg (Tab. 1). 

 Addition of ascorbic acid at the lower level augmented the response while the higher 

 supplementation effected a value similar to the control. Whether these changes in 



3^^ Europ. Symp. on Gal. Tissues 



