Isolated mitochondria from liver or brown adipose tissue of obese ob/ob mice demonstrated increased rates of Ca2+ uptake and release compared with those of lean mice. This enhanced transport activity was not found in mitochondria from kidney or skeletal muscle. Respiration-induced membrane potential was the same in mitochondria from lean and ob/ob mice. It is therefore concluded that the increased Ca2+ uptake rates reflect an activation of the Ca2+ uniporter rather than a change in the electrophoretic driving force. As mitochondria from pre-obese ob/ob mice did not show elevated rates of Ca2+ transport, the activated transport in the obese animals was thus a consequence of the state of obesity rather than being a direct effect of the ob/ob genotype. It is suggested that the enhanced activity of the Ca2+-transport pathways in liver and brown adipose tissue may alter metabolic functions in these tissues by modifying cytoplasmic or intramitochondrial Ca2+ concentrations.

The specific binding capacity for purine nucleotides in brown-adipose-tissue mitochondria is thought to indicate the capacity of the proton-conductance pathway which leads to uncoupled respiration. This functional relationship was investigated in studies measuring initial Ca2+-uptake rates and membrane potential in the presence or absence of GDP in brown-adipose-tissue mitochondria with different GDP-binding capacities. The mitochondria from pre-obese and obese ob/ob mice were less able than those from lean control mice to dissipate membrane potential in the absence of GDP. Mitochondria from the obese animals also maintained a higher Ca2+-uptake rate without GDP in comparison with the rate found with mitochondria from the lean mice. The GDP-dependence of Ca2+ uptake was greater in brown-adipose-tissue mitochondria from cold-adapted animals than in those from animals kept at 22 degrees C or at thermoneutrality (33 degrees C). It is concluded that Ca2+-uptake rate and membrane-potential values are depressed in the absence of GDP and indicate indirectly the influence of purine nucleotides on maintaining the proton electrochemical gradient in brown-adipose-tissue mitochondria. It is also apparent that the lower GDP-binding capacity in mitochondria from ob/ob mice is related to a decreased ability to dissipate the proton electrochemical gradient.

A hypothesis of Mason & Kodicek [(1970) Biochem. J. 120, 515-521] that esterified nicotinic acid in niacytin from cereals is a precursor for trigonelline was investigated in rats. Single oral doses of niacytin resulted in the excretion of trigonelline in urine but only in rats that were niacin-deficient and were fed a cereal diet. These animals were found to have an abnormally permeable intestine, which allowed the uptake of molecules not usually absorbed. Orally administered synthetic [14C]nicotinoyl[3H]methylcellulose was shown to be absorbed by niacin-deficient rats on a cereal diet and [14C]trigonelline was excreted in urine. These data indicate that dietary cereal induces a permeability defect in the intestinal mucosa of niacin-deficient rats, which allows the uptake of macromolecular niacytin. The nicotinoyl pyridine nitrogen atom is then methylated and slow hydrolysis releases trigonelline from the macromolecule.

The chemical identity of vitamin D in the egg of the domestic fowl was studied by analysing radioactivity in eggs from hens injected with [ 3 H]cholecalciferol. Labelled molecules were found throughout the egg, but the concentration of total radioactivity in albumin was only 5-7% of that in yolk. In lipid extracts of yolk, more than 90% of the radioactivity was as unchanged cholecalciferol and 5% as 25-hydroxycholecalciferol. Only about 3% of the radioactivity in albumin was chloroform-soluble, and of this 40% was 25-hydroxycholecalciferol and 15% was cholecalciferol. Evidence is presented to support the idea that the specific transport of cholecalciferol into yolk is mediated by a cholecalciferol-binding protein in blood. This protein forms a complex with yolk proteins in transit from liver to ovary via the blood. A cholecalciferol-binding protein, chromatographically similar to that from blood, was found in egg yolk. It is postulated that cholecalciferol forms part of a complex with its specific binding protein, Ca 2+ and the yolk phosphoprotein, phosvitin. This complex is then incorporated into yolk by the thecal cells of the ovarian follicle.

1. The mechanism of vitamin D esterification in the rat was studied with liver, small-intestinal mucosa, pancreatic juice and blood plasma as enzyme sources and [1− 3 H]cholecalciferol, [U− 14 C]ergocalciferol and [4− 14 C]cholesterol as substrates. 2. No esterification of vitamin D could be detected with liver preparations nor with homogenates or acetone-dried powder extracts of intestinal mucosa. 3. Pancreatic juice esterified [1− 3 H]cholecalciferol with oleic acid, and specificity studies indicated that a cholesterol-esterifying enzyme was using vitamin D as substrate. 4. Plasma cholesterol-esterifying enzyme also esterified vitamin D. 5. The specificity of the esterification reaction is discussed in relation to ( a ) the molecular structure of the substrates and ( b ) their availability, in a micellar solution, to the enzyme. 6. It is concluded that cholesterol-esterifying enzymes esterify vitamin D in vivo during absorption from the small intestine and while it is transported in blood.

1. Vitamin D-deficient rachitic rats were given [1− 3 H]cholecalciferol by gastric intubation. After 24hr., diethyl ether extracts of liver and kidney contained 5–11% and 4·5–20% respectively of total vitamin D apparently esterified with long-chain fatty acids. 2. A two-dimensional thin-layer chromatographic technique was devised that completely separated seven synthetic vitamin D esters according to the chain length and number of double bonds in the fatty acid component. When the ‘vitamin D ester’ fraction from liver or kidney was co-chromatographed with the standard esters, radioactivity appeared mainly in vitamin D palmitate, stearate, oleate and linoleate regions. The proportion of radioactivity in the saturated fatty acid esters was higher in kidney than in liver. 3. The same percentage of tissue vitamin D in the esterified form was found at each of two dosages of vitamin D. 4. The possible specificity of a vitamin D esterification mechanism is discussed.

1. The tissue contents of vitamin D alcohol and ester were estimated in rats 5, 10, 24, 48 and 72hr. after peroral administration of [1− 3 H]cholecalciferol. 2. The total vitamin D in liver decreased in an exponential fashion from 19% of the dose at 5hr. to 0·6% at 72hr., but the ester content remained at a relatively constant low value from 5hr., so that by 72hr. it represented 67% of the total vitamin D. Vitamin D ester in kidney increased slowly to 48hr., but by 72hr. it was only 10% of the total vitamin D. 3. The small intestine, unlike liver and kidney, contained a higher content of vitamin D ester 10hr. after administration than at later times, and it is postulated that some vitamin D was esterified during absorption from the alimentary tract. 4. Plasma contained vitamin D ester at all time intervals, and it is suggested that ester found in liver and kidney could have been transported to these sites in the blood. 5. Thoracic-duct lymph was found to transport 43% of a peroral dose of vitamin D in 12hr., of which 1·4% was esterified. The fatty acid components of the lymph vitamin D ester, determined by two-dimensional thinlayer chromatography, were mainly palmitate (31%), stearate (25%), oleate (16%) and linoleate (16%). This pattern was similar to that previously found in liver.