Microincineration, Electron Microscopy, and Electron Diffraction of Calcium Phosphate-Loaded Mitochondria

Abstract

Isolated rat liver mitochondria were incubated in vitro under conditions supporting the massive accumulation of calcium and phosphate. Samples were embedded, thin sectioned, and examined in the electron microscope. The intramitochondrial distribution of insoluble or structure-bound mineral substances was studied by electron microscopy coupled with recently developed techniques of high resolution microincineration. As shown previously, the ion-loaded mitochondria acquire large, internal granules which have inherent electron opacity indicative of high mineral content. Study of ash patterns in preselected areas of sections directly confirmed the high mineral content of the granules, and the appearance of the residues was consistent with the copresence in the granules of some organic material. Other mitochondrial structures were almost devoid of mineral. Thin sections of unincubated control mitochondria also were incinerated. They were found to contain appreciable amounts of intrinsic mineral, seemingly associated with membranes. The normal, dense matrix granules commonly seen in unaltered mitochondria could be seen in intact sections of these control preparations, but after burning no definite correspondence of any ash to the granules could be demonstrated. The normal granules perhaps do not contain mineral. Heating experiments on ash patterns of all the preparations demonstrated the thermal stability and crystallizability of the ash. The crystallized ash of the in vitro-produced dense granules was tentatively shown by electron diffraction to be 3-tricalcium phosphate (whitlockite). This, together with evidence from the literature, suggests that the original, noncrystalline mineral may be a colloidal, subcrystalline precursor of calcium-deficient hydroxyapatite. Experiments were performed on synthetic calcium phosphates for comparison. Other possible applications of the microincineration techniques are briefly discussed.

Electron-Microscopic Observations in Various Functional Stages of the Brown and White Adipose Tissue in the Newborn Rabbit

“…rabbits. The significant decrease of their number in brown adipose cells of rabbits fasted at low temperature cannot be as yet explained, the more so as their exact composition is unknown [25]. The above ultrastructural changes are in accordance with physiological data [2,3,20,24] that the brown adipose cells not only contain the sub strates but are also the sites of thermoregulatory heat production.…”

Amorphous calcium phosphate in casein micelles of bovine milk

“…Results and Discussion: The molar (Ca + Mg)/P i and Mg/Ca ratios of 1.66 and 0.03, respectively, in the CPC of bulk milk are similar to values reported for mitochondrial ACP (8). It has been generally assumed (9) that the vast majority of Zn in cow's milk is bound to the casein protein, but, the present data show that as much as 60% of the total milk Zn is located in the CPC giving it a Zn/Ca ratio of 0.003, which is in line with Zn levels found in the mineral phase of other calcified tissues (i0,Ii).…”

Influence of phosphorus storage on the uptake of cadmium by the marine alga Macrocystis pyrifera

“…Sections 500 nm thick were cut with a glass knife onto a water trough containing a pH 7.5 NH4HC03 buffer. Phosphate mineral components in cells can solubilize rapidly in the water trough during sectioning (the pH of bidistilled water is 6 or less), but are stable above pH 7 (Thomas and Greenawalt 1968).…”

“…The concentrations of mineral components in the cells were, therefore, enhanced for better sensitivity by microincineration of thick tissue sections (Thomas and Greenawalt 1968;Adamec et al 1979). This method removes the organic components, thus reducing a thick section to a thin one comprising only the concentrated mineral remnants of the cell.…”