Hip fractures represent a serious health risk in the elderly, with significant associated morbidity and mortality. There is now an emerging literature that suggests that chronic hyponatremia increases the adjusted odds ratio (OR) for both falls and fractures in the elderly. Hyponatremia appears to contribute to falls and fractures by two mechanisms: (i) it produces mild cognitive impairment resulting in unsteady gait and falls and (ii) it directly contributes to osteoporosis and increased bone fragility by inducing increased bone resorption to mobilize sodium. There is debate over the effect of hyponatremia on the production of osteoporosis, as one study found decreased bone mineral density (BMD) and another did not. Should we be screening for low serum sodium in patients with osteoporosis or assessing BMD in patients with hyponatremia? The final answer is yet to come from prospective studies that allocate elderly individuals with mild hyponatremia to receive active treatment or not for hyponatremia and see if this intervention prevents gait disturbances and changes in BMD reducing fracture risk. In the meantime, physicians caring for elderly patients must be aware of the association between hyponatremia and bone problems. As serum sodium is a readily available, simple and affordable biochemical measurement, clinicians should look for hyponatremia in elderly patients who take medications that can cause hyponatremia. Also, elderly patients with unsteady gait and/or confusion should be checked for the presence of mild hyponatremia and if present it should not be ignored. Finally, elderly patients presenting with an orthopedic injury should have serum sodium checked and corrected if hyponatremia is present.

It has been previously shown that intestinal proteases translocate into the circulation during hemorrhagic shock and contribute to proteolysis in distal organs. However, consequences of this phenomenon have not previously been investigated using high-throughput approaches. Here, a shotgun label-free quantitative proteomic approach was utilized to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats to verify the possible role of uncontrolled proteolytic activity in shock. Plasma was collected from rats after hemorrhagic shock (HS) consisting of 2-h hypovolemia followed by 2-h reperfusion, and from healthy control (CTRL) rats. A new two-step enrichment method was applied to selectively extract peptides and low molecular weight proteins from plasma, and directly analyze these samples by tandem mass spectrometry. One hundred twenty-six circulating peptides were identified in CTRL and 295 in HS animals. Ninety-six peptides were present in both conditions; of these, 57 increased and 30 decreased in shock. In total, 256 peptides were increased or present only in HS confirming a general increase in proteolytic activity in shock. Analysis of the proteases that potentially generated the identified peptides suggests that the larger relative contribution to the proteolytic activity in shock is due to chymotryptic-like proteases. These results provide quantitative confirmation that extensive, system-wide proteolysis is part of the complex pathologic phenomena occurring in hemorrhagic shock.

A preliminary mass spectrometry based shotgun protocol was set up to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats with the aim of verifying the possible role of uncontrolled proteolytic activity in circulatory shock. Since the hemorrhagic shock model requires heparin as anticoagulant, a preliminary experiment using plasma sample obtained in the presence/absence of heparin from healthy rats was performed to determine whether its presence is fully compatible with the peptidomic protocol proposed. The entire protocol was tested in a pilot experiment to compare the peptidome of healthy or heparin-anticoagulated rats subjected to hemorrhagic shock.

Background

Hip fractures are among the most serious bone fractures in the elderly, producing significant morbidity and mortality. Several observational studies have found that mild hyponatremia can adversely affect bone, with fractures occurring as a potential complication. We examined if there is an independent association between prolonged chronic hyponatremia (>90 days duration) and risk of hip fracture in the elderly.

Methods

We performed a retrospective cohort study in adults >60 years of age from a prepaid health maintenance organization who had two or more measurements of plasma sodium between 2005 and 2012. The incidence of hip fractures was assessed in a very restrictive population: subjects with prolonged chronic hyponatremia, defined as plasma sodium values <135 mmol/L, lasting >90 days. Multivariable Cox regression was performed to determine the hazard ratio (HR) for hip fracture risk associated with prolonged chronic hyponatremia after adjustment for the propensity to have hyponatremia, fracture risk factors and relevant baseline characteristics.

Results

Among 31 527 eligible patients, only 228 (0.9%) had prolonged chronic hyponatremia. Mean plasma sodium was 132 ± 5 mmol/L in hyponatremic patients and 139 ± 3 mmol/L in normonatremic patients (P < 0.001). The absolute risk for hip fracture was 7/282 in patients with prolonged chronic hyponatremia and 411/313 299 in normonatremic patients. Hyponatremic patients had a substantially elevated rate of hip fracture [adjusted HR 4.52 (95% CI 2.14-9.6)], which was even higher in those with moderate hyponatremia (<130 mmol/L) [adjusted HR 7.61 (95% CI 2.8-20.5)].

Conclusion

Mild prolonged chronic hyponatremia is independently associated with hip fracture risk in the elderly population, although the absolute risk is low. However, proof that correcting hyponatremia will result in a reduction of hip fractures is lacking.

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.