Spinal cord from body donors is suitable for multicolor immunofluorescence

Immunohistochemistry is a powerful tool for studying neuronal tissue from humans at the molecular level. Obtaining fresh neuronal tissue from human organ donors is difficult and sometimes impossible. In anatomical body donations, neuronal tissue is dedicated to research purposes and because of its easier availability, it may be an alternative source for research. In this study, we harvested spinal cord from a single organ donor 2 h (h) postmortem and spinal cord from body donors 24, 48, and 72 h postmortem and tested how long after death, valid multi-color immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry is possible. We used general and specific neuronal markers and glial markers for immunolabeling experiments. Here we showed that it is possible to visualize molecularly different neuronal elements with high precision in the body donor spinal cord 24 h postmortem and the quality of the image data was comparable to those from the fresh organ donor spinal cord. High-contrast multicolor images of the 24-h spinal cords allowed accurate automated quantification of different neuronal elements in the same sample. Although there was antibody-specific signal reduction over postmortem intervals, the signal quality for most antibodies was acceptable at 48 h but no longer at 72 h postmortem. In conclusion, our study has defined a postmortem time window of more than 24 h during which valid immunohistochemical information can be obtained from the body donor spinal cord. Due to the easier availability, neuronal tissue from body donors is an alternative source for basic and clinical research

Intended and unintended consequences: Changes in opioid prescribing practices following two policies in North Carolina, 2012–2018 – A controlled interrupted time series analysis

Poster presented at the 38th International Conference on Pharmacoepidemiology & Therapeutic Risk Management. Objective: To understand the extent to which unintended prescribing consequences followed implementation of two statewide opioid prescribing policies among privately insured, opioid-naïve individuals in North Carolina between 2012 and 2018

Bone resorption stimulated by elevated serum 1,25-(OH)2-vitamin D concentrations in healthy men

Bone resorption stimulated by elevated serum 1,25-(OH)2-vitamin D concentrations in healthy men. We evaluated whether calcitriol administration to healthy men stimulates bone resorption. We compared serum 1,25-(OH)2-D concentrations, Ca and PO4 balances, and urinary hydroxyproline excretion rates in four healthy men adapted to a low Ca diet providing only 4.0 ± 0.2 SD mmoles Ca/day to those in four healthy men eating a comparable diet (4.2 ± 0.9 mmoles Ca/day) during the chronic oral administration of calcitriol, 0.75 µg every 6hr. Serum 1,25-(OH)2-D levels averaged 94 ± 16 pM during the control studies and 209 ± 35 pM during calcitriol administration. Net intestinal Ca absorption averaged 0.5 ± 0.3 mmoles/day during control and 1.8 ± 0.5 mmoles/day during calcitriol (P < 0.005), but urinary Ca excretion averaged 8.7 ± 2.0 mmoles/day during calcitriol as compared to 2.9 ± 1.4 mmoles/day during control (P < 0.005). Thus, mean Ca balance, which averaged -2.4 ± 1.2 mmoles/day during control, was more negative during calcitriol at -6.3 ± 2.4 mmoles/day (P < 0.05). Average daily PO4 balances averaged +7.7 ± 1.5 mmoles/day during control but only tended to be negative during calcitriol at -1.1 ± 5.4 mmoles/day, (NS). Urinary hydroxyproline excretion averaged 0.26 ± 0.03 mmoles/day during control and 0.49 ± 0.06 during calcitriol (P < 0.001). We conclude that elevated serum 1,25-(OH)2-D concentrations in healthy men eating low Ca diets stimulate bone resorption.La résorption osseuse stimulé par des concentrations élevées de 1,25-(OH)2-vitamine D chez des hommes normaux. Nous avons recherché si l'administration de calcitriol à des hommes normaux stimule la résorption osseuse. Nous avons comparé les concentrations sériques de 1,25-(OH)2-D, les balances de Ca et PO4 et l'excrétion urinaire d'hydroxyproline chez quatre hommes normaux adaptés à un régime pauvre en Ca apportant seulement 4,0 ± 0,2 SD mmoles Ca/jour à ceux de quatre hommes normaux consommant un régime comparable (4,2 ± 0,9 mmoles Ca/jour) pendant l'administration orale chronique de calcitriol, 0,75 µg toutes les 6 heures. Les concentrations sériques de 1,25-(OH)2-D étaient en moyenne de 94 ± 16 pM pendant les études contrôles et de 209 ± 35 pM pendant l'administration de calcitriol. L'absorption intestinale nette de Ca était en moyenne de 0,5 ± 0,3 mmoles/jour pendant le contrôle et de 1,8 ± 0,5 mmoles/jour pendant la prise de calcitriol (P < 0,005), mais l'excrétion urinaire de Ca était en moyenne de 8,7 ± 2,0 mmoles/jour pendant le calcitriol, par rapport à 2,9 ± 1,4 mmoles/jour pendant le contrôle (P < 0,005). Ainsi, la balance moyenne de Ca, qui était en moyenne de -2,4 ± 1,2 mmoles/jour pendant le contrôle, était encore plus négative pendant le calcitriol, de -6,3 ± 2,4 mmoles/jour (P < 0,05). Les balances moyennes journalières de PO4 étaient de +7,7 ± 1,5 mmoles/jour pendant le contrôle, mais n'avaient que simplement tendance à être négatives pendant le calcitriol à -1,1 ± 5,4 mmoles/jour, (NS). L'excrétion urinaire d'hydroxyproline était de 0,26 ± 0,03 mmoles/jour pendant le contrôle et de 0,49 ± 0,06 pendant le calcitriol (P < 0,001). Nous concluons que les concentrations élevées de 1,25-(OH)2-D sérique chez des hommes normaux consommant des régimes pauvres en calcium stimulent la résorption osseuse

Hydrochlorothiazide inhibits bone resorption in men despite experimentally elevated serum 1,25-dihydroxyvitamin D concentrations

Hydrochlorothiazide inhibits bone resorption in men despite experimentally elevated serum 1,25-dihydroxy vitamin D concentrations. We evaluated the effects of hydrochlorothiazide administration in relation to Ca balance, the PTH and vitamin D endocrine systems, acid-base balance, and bone. We studied six healthy men fed constant diets providing only 5.1 ± 0.7 SD mmoles Ca/day. Three of the men were also given calcitriol, 0.5 µg 6-hrly throughout their studies. All subjects were observed during 18 control days and then during 18 days of hydrochlorothiazide (HTZ) administration, 25mg 12-hrly. Observations during control days 11 through 16 were compared to those during days 7 through 18 of HTZ administration, inclusively. Directional changes during HTZ did not differ among subjects not given or given calcitriol. For all six subjects, control net intestinal Ca absorption, serum 1,25-(OH)2-D concentrations, serum iPTH concentrations, and daily urine cAMP excretion averaged 0.5 ± 2.2 mmoles/day, 162 ± 51pM, 4.3 ± 2.2 µl Eq/ml and 4.2 ± 0.9 µmoles/day, respectively; none changed during HTZ. As expected, HTZ administration was accompanied by a fall in urinary Ca excretion, averaging - 1.4 ± 0.8 mmoles/day; P < 0.01. HTZ administration was also accompanied by less negative Ca balances, averaging + 1.6 ± 1.0 mmoles/day; P < 0.025, and by a fall in daily urinary hydroxy proline excretion averaging -0.13 P < 0.09 mmoles/day; P < 0.025. We interpret these data to indicate that HTZ administration is accompanied by an inhibition of bone resorption. HTZ administration also raised serum HCO3 concentrations by + 2.7 ± 0.5 mEq/liter; P < 0.001 and blood pH by + 0.05 ± 0.02 units; P < 0.005. Since HTZ administration did not change either serum iPTH or 1,25-(OH)2-D concentrations nor urinary cAMP excretion, inhibition of bone resorption may be mediated by either relative alkalosis, a reduced skeletal sensitivity to PTH or 1,25-(OH)2-D, or, possibly, by a direct effect of HTZ on bone

The importance of renal net acid excretion as a determinant of fasting urinary calcium excretion

The importance of renal net acid excretion as a determinant of fasting urinary calcium excretion. To evaluate the effects of changing rates of fixed acid production on fasting urine Ca/creatinine, we studied five healthy men fed constant diets during control conditions (serum HC03 27.3 ± 2.6 SD mEq/liter and blood H+ 40.4 ± 1.5 µEq/liter) and then during the administration of NH4C1 3.0 mEq/kg/day (serum HC03 22.5 ± 4.9 mEq/liter; P < 0.025, and H+ 46.8 ± 2.3 mEq/liter; P < 0.005). In addition to the expected increase in daily urinary Ca excretion from 5.2 ± 2.0 to 12.5 ± 3.0 mmole/day; P < 0.001 as daily urinary net acid excretion was increased from 48 ± 32 to 257 ± 33 mEq/day; P < 0.001 we observed that fasting urinary net acid/creatinine excretion also increased from 2.9 ± 1.2 to 11.1 ± 1.2 mEq/mmole creatinine; P < 0.001 and fasting urine Ca/creatinine increased from 0.158 ± 0.111 to 0.456 ± 0.109 mmole/mmole creatinine; P < 0.005. The additional Ca appearing in the urine during acidosis ultimately reflected augmented net bone resorption since daily urinary hydroxyproline excretion was increased from 0.232 ± 0.062 to 0.377 ± 0.108 mmole/day; P < 0.01. Since variations in diet composition can cause fixed acid production and thus renal net acid excretion to vary from about zero to 200 mEq/day, such a range could cause fasting Ca/creatinine to vary from 0.09 to 0.37 mmole/mmole (0.03 to 0.13 mg/mg) and should be taken into account in the evaluation of fasting Ca/creatinine

Dietary calcium and serum 1,25-(OH)2-vitamin D concentrations as determinants of calcium balance in healthy men

Dietary calcium and serum 1,25-(OH)2-vitamin D concentrations as determinants of calcium balance in healthy men. We previously reported that experimental elevations of serum 1,25-(OH)2-vitamin D [1,25-(OH)2-D] concentrations produced by the chronic oral administration of calcitriol, 0.75 µg every 6 hr, to healthy human males eating diets providing only 4 mmoles Ca/day stimulate net bone resorption as evidenced by more negative Ca balances and higher rates of urinary hydroxyproline excretion. To determine whether increased dietary Ca intake modifies this response we have compared serum 1,25-(OH)2-D and iPTH concentrations, Ca and PO4 balances, and urinary hydroxyproline excretion in three healthy human males adapted to diets providing 22.3 ± 1.3 mmoles Ca/day and three healthy human males adapted to diets providing 9.3 ± 0.7 mmoles Ca/day before and during the continuous oral administration of calcitriol 0,5 µg every 6hr. For all six subjects, serum 1,25-(OH)2-D levels averaged 89 ± 25 pM during control and 143 ± 26 pM during calcitriol. Net intestinal Ca absorption and urinary Ca excretion rose during calcitriol but Ca balances did not change, averaging +2.2 ± 2.2 mmoles/day during control and +4.3 ± 2,2 mmoles/day during calcitriol for the subjects fed 22 mmoles Ca/day and - 1.6 ± 1.5 mmoles Ca/day during control and - 1.7 ± 2.0 mmoles Ca/day during calcitriol for the subjects fed 9 mmoles Ca/day. Urinary hydroxyproline excretion also did not change. Thus, when serum 1,25-(OH)2-D levels are elevated, the availability of dietary Ca appears to prevent more negative Ca balances and increased urinary hydroxyproline excretion suggesting that net bone resorption is not stimulated.Le calcium alimentaire et les concentrations sériques de 1,25-(OH)2-vitamine D déterminants de la balance calcique chez des hommes sains. Nous avons préalablement rapporté que des élévations expérimentales des concentrations sériques de 1,25-(OH)2-D par administration chronique orale de calcitriol, 0,75 µg toutes les 6 heures à des hommes sains ingérant des régimes n'apportant que 4 mmoles Ca/jour stimulent la résorption osseuse nette, ce qui est mis en évidence par des balances Ca plus négatives et par des débits d'excrétion urinaire d'hydroxyproline plus élevés. Afin de déterminer si une augmentation de l'apport en Ca alimentaire modifie cette réponse, nous avons comparé les concentrations sériques de 1,25-(OH)2-D et d'iPTH, les balances Ca et PO4 et l'excrétion urinaire d'hydroxyproline chez trois hommes sains adaptés à des régimes apportant 22,3 ± 1,3 mmoles Ca/jour et chez trois hommes sains adaptés à des régimes apportant 9,3 ± 0,7 mmoles Ca/jour avant et pendant l'administration orale continue de calcitriol, 0,5 µg toutes les 6 heures. Pour les six sujets, les niveaux sériques de 1,25-(OH)2-D étaient en moyenne de 89 ± 25 pM pendant le contrôle et de 143 ± 26 pM pendant le calcitriol. L'absorption intestinale nette de Ca et l'excrétion urinaire de Ca se sont élevées pendant le calcitriol, mais les balances Ca n'ont pas changé, étant en moyenne de +2,2 ± 2,2 mmoles/jour pendant le contrôle et de 4,3 ± 2,2 mmoles/jour pendant le calcitriol chez les sujets ingérant 22 mmoles Ca/jour et de - 1,6 ± 1,5 mmoles Ca/jour pendant le contrôle et de 1,7 ± 2,0 mmoles Ca/jour pendant le calcitriol chez les sujets ingérant 9 mmoles Ca/jour. L'hydroxyproline urinaire aussi ne s'est pas modifiée. Ainsi, lorsque les niveaux sériques de 1,25-(OH)2-D sont élevés, la disponibilité du calcium alimentaire parait prévenir des balances Ca plus négatives et une excrétion urinaire d'hydroxyproline élevée, suggérant que la résorption osseuse nette n'est pas stimulée