Targeted metabolomics reveals plasma biomarkers and metabolic alterations of the aging process in healthy young and older adults.

With the exponential growth in the older population in the coming years, many studies have aimed to further investigate potential biomarkers associated with the aging process and its incumbent morbidities. Age is the largest risk factor for chronic disease, likely due to younger individuals possessing more competent adaptive metabolic networks that result in overall health and homeostasis. With aging, physiological alterations occur throughout the metabolic system that contribute to functional decline. In this cross-sectional analysis, a targeted metabolomic approach was applied to investigate the plasma metabolome of young (21-40y; n = 75) and older adults (65y + ; n = 76). A corrected general linear model (GLM) was generated, with covariates of gender, BMI, and chronic condition score (CCS), to compare the metabolome of the two populations. Among the 109 targeted metabolites, those associated with impaired fatty acid metabolism in the older population were found to be most significant: palmitic acid (p < 0.001), 3-hexenedioic acid (p < 0.001), stearic acid (p = 0.005), and decanoylcarnitine (p = 0.036). Derivatives of amino acid metabolism, 1-methlyhistidine (p = 0.035) and methylhistamine (p = 0.027), were found to be increased in the younger population and several novel metabolites were identified, such as cadaverine (p = 0.034) and 4-ethylbenzoic acid (p = 0.029). Principal component analysis was conducted and highlighted a shift in the metabolome for both groups. Receiver operating characteristic analyses of partial least squares-discriminant analysis models showed the candidate markers to be more powerful indicators of age than chronic disease. Pathway and enrichment analyses uncovered several pathways and enzymes predicted to underlie the aging process, and an integrated hypothesis describing functional characteristics of the aging process was synthesized. Compared to older participants, the young group displayed greater abundance of metabolites related to lipid and nucleotide synthesis; older participants displayed decreased fatty acid oxidation and reduced tryptophan metabolism, relative to the young group. As a result, we offer a better understanding of the aging metabolome and potentially reveal new biomarkers and predicted mechanisms for future study.

A Metabolomic Analysis of the Sex-Dependent Hispanic Paradox.

In Mexican Americans, metabolic conditions, such as obesity and type 2 diabetes (T2DM), are not necessarily associated with an increase in mortality; this is the so-called Hispanic paradox. In this cross-sectional analysis, we used a metabolomic analysis to look at the mechanisms behind the Hispanic paradox. To do this, we examined dietary intake and body mass index (BMI; kg/m2) in men and women and their effects on serum metabolomic fingerprints in 70 Mexican Americans (26 men, 44 women). Although having different BMI values, the participants had many similar anthropometric and biochemical parameters, such as systolic and diastolic blood pressure, total cholesterol, and LDL cholesterol, which supported the paradox in these subjects. Plasma metabolomic phenotypes were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). A two-way ANOVA assessing sex, BMI, and the metabolome revealed 23 significant metabolites, such as 2-pyrrolidinone (p = 0.007), TMAO (p = 0.014), 2-aminoadipic acid (p = 0.019), and kynurenine (p = 0.032). Pathway and enrichment analyses discovered several significant metabolic pathways between men and women, including lysine degradation, tyrosine metabolism, and branch-chained amino acid (BCAA) degradation and biosynthesis. A log-transformed OPLS-DA model was employed and demonstrated a difference due to BMI in the metabolomes of both sexes. When stratified for caloric intake (<2200 kcal/d vs. >2200 kcal/d), a separate OPLS-DA model showed clear separation in men, while females remained relatively unchanged. After accounting for caloric intake and BMI status, the female metabolome showed substantial resistance to alteration. Therefore, we provide a better understanding of the Mexican-American metabolome, which may help demonstrate how this population-particularly women-possesses a longer life expectancy despite several comorbidities, and reveal the underlying mechanisms of the Hispanic paradox.

Enhanced Detection of Short-Chain Fatty Acids Using Gas Chromatography Mass Spectrometry.

Short-chain fatty acids (SCFAs) are produced mainly by intestinal microbiota and play an important role in many host biological processes such as immune system development, glucose and energy homeostasis, and regulation of immune response and inflammation. In addition, they participate in the regulation of anorectic hormones, which have a role in appetite control, tumor suppression, and regulating the central and peripheral nervous systems. As such, there is great interest in monitoring levels of SCFAs in various biological samples. Due to the highly hydrophilic and volatile characteristics of SCFAs, optimizing extraction and sample preparation procedures is often a central component to further improve SCFA quantification. Here, we describe a rapid and highly sensitive analytical method for measuring SCFAs in human serum and feces. Briefly, SCFAs are protected by adding sodium hydroxide, followed by a one-step extraction (pH > 7). Then, SCFAs are quantified by gas chromatography coupled to mass spectrometry (GC-MS) after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). This method demonstrates excellent sensitivity, linearity, and derivatization efficiency for simultaneous determination of 14 different SCFAs. Further, this validated method can be successfully applied to quantify SCFAs in micro-scale biological samples. In summary, we describe efficient and advanced sample preparation and detection procedures that are critically needed for monitoring SCFA concentrations in human biological samples. © 2021 Wiley Periodicals LLC. Basic Protocol: SCFA extraction and detection from fecal and serum samples with gas chromatography-mass spectrometry.

Metabolomics Analysis of Viral Therapeutics.

Virotherapy, enabled by recent advances in the transdisciplinary field of biotechnology, has emerged as a powerful tool for use in anticancer treatment, gene therapy, immunotherapy, etc. Examining the effects of viruses and virus-derived immune-modulating therapeutics is of great fundamental and clinical interest. Here we describe a sample preparation protocol for metabolite extraction from virus-infected tissue, in addition to liquid chromatography-mass spectrometry conditions essential for subsequent analysis. This metabolomics approach delivers highly sensitive and specific metabolite information on various biospecimens. Such an approach may be adopted to monitor biological changes in over 30 relevant metabolic pathways in response to viral infection and also viral therapeutics.

Ankle and midtarsal joint quasi-stiffness during walking with added mass.

Examination of how the ankle and midtarsal joints modulate stiffness in response to increased force demand will aid understanding of overall limb function and inform the development of bio-inspired assistive and robotic devices. The purpose of this study is to identify how ankle and midtarsal joint quasi-stiffness are affected by added body mass during over-ground walking. Healthy participants walked barefoot over-ground at 1.25 m/s wearing a weighted vest with 0%, 15% and 30% additional body mass. The effect of added mass was investigated on ankle and midtarsal joint range of motion (ROM), peak moment and quasi-stiffness. Joint quasi-stiffness was broken into two phases, dorsiflexion (DF) and plantarflexion (PF), representing approximately linear regions of their moment-angle curve. Added mass significantly increased ankle joint quasi-stiffness in DF (p < 0.001) and PF (p < 0.001), as well as midtarsal joint quasi-stiffness in DF (p < 0.006) and PF (p < 0.001). Notably, the midtarsal joint quasi-stiffness during DF was ~2.5 times higher than that of the ankle joint. The increase in midtarsal quasi-stiffness when walking with added mass could not be explained by the windlass mechanism, as the ROM of the metatarsophalangeal joints was not correlated with midtarsal joint quasi-stiffness (r = -0.142, p = 0.540). The likely source for the quasi-stiffness modulation may be from active foot muscles, however, future research is needed to confirm which anatomical structures (passive or active) contribute to the overall joint quasi-stiffness across locomotor tasks.

Differences in unilateral chest press muscle activation and kinematics on a stable versus unstable surface while holding one versus two dumbbells.

Training the bench press exercise on a traditional flat bench does not induce a level of instability as seen in sport movements and activities of daily living. Twenty participants were recruited to test two forms of instability: using one dumbbell rather than two and lifting on the COR bench compared to a flat bench. Electromyography (EMG) amplitudes of the pectoralis major, middle trapezius, external oblique, and internal oblique were recorded and compared. Differences in range of motion (ROM) were evaluated by measuring an angular representation of the shoulder complex. Four separate conditions of unilateral bench press were tested while lifting on a: flat bench with one dumbbell, flat bench with two dumbbells, COR Bench with one dumbbell, and COR Bench with two dumbbells. The results imply that there are no differences in EMG amplitude or ROM between the COR bench and traditional bench. However, greater ROM was found to be utilized in the single dumbbell condition, both in the COR bench and the flat bench.

Dextrose and morrhuate sodium injections (prolotherapy) for knee osteoarthritis: a prospective open-label trial.

OBJECTIVES: This study determined whether injection with hypertonic dextrose and morrhuate sodium (prolotherapy) using a pragmatic, clinically determined injection schedule for knee osteoarthritis (KOA) results in improved knee pain, function, and stiffness compared to baseline status. DESIGN: This was a prospective three-arm uncontrolled study with 1-year follow-up. SETTING: The setting was outpatient. PARTICIPANTS: The participants were 38 adults who had at least 3 months of symptomatic KOA and who were in the control groups of a prior prolotherapy randomized controlled trial (RCT) (Prior-Control), were ineligible for the RCT (Prior-Ineligible), or were eligible but declined the RCT (Prior-Declined). INTERVENTION: The injection sessions at occurred at 1, 5, and 9 weeks with as-needed treatment at weeks 13 and 17. Extra-articular injections of 15% dextrose and 5% morrhuate sodium were done at peri-articular tendon and ligament insertions. A single intra-articular injection of 6 mL 25% dextrose was performed through an inferomedial approach. OUTCOME MEASURES: The primary outcome measure was the validated Western Ontario McMaster University Osteoarthritis Index (WOMAC). The secondary outcome measure was the Knee Pain Scale and postprocedure opioid medication use and participant satisfaction. RESULTS: The Prior-Declined group reported the most severe baseline WOMAC score (p=0.02). Compared to baseline status, participants in the Prior-Control group reported a score change of 12.4±3.5 points (19.5%, p=0.002). Prior-Decline and Prior-Ineligible groups improved by 19.4±7.0 (42.9%, p=0.05) and 17.8±3.9 (28.4%, p=0.008) points, respectively; 55.6% of Prior-Control, 75% of Prior-Decline, and 50% of Prior-Ineligible participants reported score improvement in excess of the 12-point minimal clinical important difference on the WOMAC measure. Postprocedure opioid medication resulted in rapid diminution of prolotherapy injection pain. Satisfaction was high and there were no adverse events. CONCLUSIONS: Prolotherapy using dextrose and morrhuate sodium injections for participants with mild-to-severe KOA resulted in safe, significant, sustained improvement of WOMAC-based knee pain, function, and stiffness scores compared to baseline status.

Association between disease-specific quality of life and magnetic resonance imaging outcomes in a clinical trial of prolotherapy for knee osteoarthritis.

OBJECTIVE: To assess the relation between knee osteoarthritis (KOA)-specific quality of life (QOL) and intra-articular cartilage volume (CV) in participants treated with prolotherapy. KOA is characterized by CV loss and multifactorial pain. Prolotherapy is an injection therapy reported to improve KOA-related QOL to a greater extent than blinded saline injections and at-home exercise, but its mechanism of action is unclear. DESIGN: Two-arm (prolotherapy, control), partially blinded, controlled trial. SETTING: Outpatient. PARTICIPANTS: Adults with ≥3 months of symptomatic KOA (N=37). INTERVENTIONS: Prolotherapy: 5 monthly injection sessions; CONTROL: blinded saline injections or at-home exercise. MAIN OUTCOME MEASURES: Primary: KOA-specific QOL scores (baseline, 5, 9, 12, 26, and 52wk; Western Ontario and McMaster University Osteoarthritis Index). Secondary: KOA-specific pain, stiffness, function (Western Ontario McMaster University Osteoarthritis Index subscales), and magnetic resonance imaging-assessed CV (baseline, 52wk). RESULTS: Knee-specific QOL improvement among prolotherapy participants exceeded that among controls (17.6±3.2 points vs 8.6±5.0 points; P=.05) at 52 weeks. Both groups lost CV over time (P<.05); no between-group differences were noted (P=.98). While prolotherapy participants lost CV at varying rates, those who lost the least CV ("stable CV") had the greatest improvement in pain scores. Among prolotherapy participants, but not control participants, the change in CV and the change in pain (but not stiffness or function) scores were correlated; each 1% CV loss was associated with 2.7% less improvement in pain score (P<.05). CONCLUSIONS: Prolotherapy resulted in safe, substantial improvement in KOA-specific QOL compared with control over 52 weeks. Among prolotherapy participants, but not controls, magnetic resonance imaging-assessed CV change (CV stability) predicted pain severity score change, suggesting that prolotherapy may have a pain-specific disease-modifying effect. Further research is warranted.

Dextrose prolotherapy for knee osteoarthritis: a randomized controlled trial.

PURPOSE: Knee osteoarthritis is a common, debilitating chronic disease. Prolotherapy is an injection therapy for chronic musculoskeletal pain. We conducted a 3-arm, blinded (injector, assessor, injection group participants), randomized controlled trial to assess the efficacy of prolotherapy for knee osteoarthritis. METHODS: Ninety adults with at least 3 months of painful knee osteoarthritis were randomized to blinded injection (dextrose prolotherapy or saline) or at-home exercise. Extra- and intra-articular injections were done at 1, 5, and 9 weeks with as-needed additional treatments at weeks 13 and 17. Exercise participants received an exercise manual and in-person instruction. Outcome measures included a composite score on the Western Ontario McMaster University Osteoarthritis Index (WOMAC; 100 points); knee pain scale (KPS; individual knee), post-procedure opioid medication use, and participant satisfaction. Intention-to-treat analysis using analysis of variance was used. RESULTS: No baseline differences existed between groups. All groups reported improved composite WOMAC scores compared with baseline status (P <.01) at 52 weeks. Adjusted for sex, age, and body mass index, WOMAC scores for patients receiving dextrose prolotherapy improved more (P <.05) at 52 weeks than did scores for patients receiving saline and exercise (score change: 15.3 ± 3.5 vs 7.6 ± 3.4, and 8.2 ± 3.3 points, respectively) and exceeded the WOMAC-based minimal clinically important difference. Individual knee pain scores also improved more in the prolotherapy group (P = .05). Use of prescribed postprocedure opioid medication resulted in rapid diminution of injection-related pain. Satisfaction with prolotherapy was high. There were no adverse events. CONCLUSIONS: Prolotherapy resulted in clinically meaningful sustained improvement of pain, function, and stiffness scores for knee osteoarthritis compared with blinded saline injections and at-home exercises.

Hypertonic dextrose injections (prolotherapy) for knee osteoarthritis: results of a single-arm uncontrolled study with 1-year follow-up.

OBJECTIVE: The objective of this study was to determine whether prolotherapy, an injection-based complementary treatment for chronic musculoskeletal conditions, improves pain, stiffness, and function in adults with symptomatic knee osteoarthritis (KOA) compared to baseline status. DESIGN: This was a prospective, uncontrolled study with 1-year follow-up. SETTING: The study was conducted in an outpatient setting. PARTICIPANTS: Adults with at least 3 months of symptomatic KOA, recruited from clinical and community settings, participated in the study. INTERVENTIONS: Participants received extra-articular injections of 15% dextrose and intra-articular prolotherapy injections of 25% dextrose at 1, 5, and 9 weeks, with as-needed treatments at weeks 13 and 17. OUTCOME MEASURES: Primary outcome measure was the validated Western Ontario McMaster University Osteoarthritis Index (WOMAC). Secondary outcome measure was the validated Knee Pain Scale (KPS). Tertiary outcome measure was procedure-related pain severity and participant satisfaction. RESULTS: Thirty-six (36) participants (60 ± 8.7 years old, 21 female) with moderate-to-severe KOA received an average of 4.3 ± 0.7 prolotherapy injection sessions over a 17-week treatment period and reported progressively improved scores during the 52-week study on WOMAC and KPS measures. Participants reported overall WOMAC score improvement 4 weeks after the first injection session (7.6 ± 2.4 points, 17.2%), and continued to improve through the 52-week follow-up (15.9 ± 2.5 points, p<0.001, 36.1%). KPS scores improved in both injected (p<0.001) and uninjected knees (p<0.05). Prescribed low-dose opioid analgesia effectively treated procedure-related pain. Satisfaction was high and there were no adverse events. Female gender, age 46-65 years old, and body-mass index of 25 kg/m(2) or less were associated with greater improvement on the WOMAC instrument. CONCLUSIONS: In adults with moderate to severe KOA, dextrose prolotherapy may result in safe, significant, sustained improvement of knee pain, function, and stiffness scores. Randomized multidisciplinary effectiveness trials including evaluation of potential disease modification are warranted to further assess the effects of prolotherapy for KOA.

Exertional paraspinal muscle rhabdomyolysis and compartment syndrome: a cause of back pain not to be missed.

Exertional compartment syndrome has been described and implicated in pain syndromes involving several fascial compartments. This entity is classically characterized in the legs, feet and forearms of athletes. We describe a case of acute, severe exertional compartment syndrome of the paraspinal muscles in a young and healthy male ultimately resulting in significant rhabdomyolysis and acute kidney injury. The rarity of the syndrome has prevented the establishment of specific guidelines for management; therefore, we will discuss this case in the context of similar previously reported cases, contrasting the various treatment approaches and outcomes described in previous reports. This discussion outlines a syndrome not commonly considered in the differential diagnosis of back pain.

Response of knee ligaments to prolotherapy in a rat injury model.

BACKGROUND: Prolotherapy is an alternative therapy for chronic musculoskeletal injury including joint laxity. The commonly used injectant, D-glucose (dextrose), is hypothesized to improve ligament mechanics and decrease pain through an inflammatory mechanism. No study has investigated the mechanical effects of prolotherapy on stretch-injured ligaments. HYPOTHESES: Dextrose injections will enlarge cross-sectional area, decrease laxity, strengthen, and stiffen stretch-injured medial collateral ligaments (MCLs) compared with controls. Dextrose prolotherapy will increase collagen fibril diameter and density of stretch-injured MCLs. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty-four rats were bilaterally MCL stretch-injured, and the induced laxity was measured. After 2 weeks, 32 MCLs were injected twice, 1 week apart, with either dextrose or saline control; 16 MCLs received no injection. Seven uninjured rats (14 MCLs) were additional controls. Two weeks after the second injection, ligament laxity, mechanical properties (n = 8), and collagen fibril diameter and density (n = 3) were assessed. RESULTS: The injury model created consistent ligament laxity (P < .05) that was not altered by dextrose injections. Cross-sectional area of dextrose-injected MCLs was increased 30% and 90% compared with saline and uninjured controls, respectively (P < .05). Collagen fibril diameter and density were decreased in injured ligaments compared with uninjured controls (P < .05), but collagen fibril characteristics were not different between injured groups. CONCLUSION: Dextrose injections increased the cross-sectional area of MCLs compared with saline-injected and uninjured controls. Dextrose injections did not alter other measured properties in this model. CLINICAL RELEVANCE: Our results suggest that clinical improvement from prolotherapy may not result from direct effects on ligament biomechanics.

Early inflammatory response of knee ligaments to prolotherapy in a rat model.

Prolotherapy is an alternative injection-based therapy for chronic musculoskeletal pain. Three different proliferants, D-glucose (dextrose), phenol-glucose-glycerine (P2G), and sodium morrhuate, used in prolotherapy are hypothesized to strengthen and reorganize chronically injured soft tissue and decrease pain through modulation of the inflammatory process. Our hypothesis is that commonly used prolotherapy solutions will induce inflammation (leukocyte and macrophage infiltration) in medial collateral ligaments (MCLs) compared to needlestick, saline injection, and no-injection controls. MCLs of 84 Sprague- Dawley rats were injected one time at both the tibial and femoral insertions. Immunohistochemistry (IHC) was used to determine the inflammatory response at three locations (tibial and femoral insertions and midsubstance) 6, 24, and 72 h after dextrose injection compared to saline- and no-injection controls and collagenase (positive control) (n = 4). qPCR was used to analyze gene expression 24 h postinjection (n = 4). Sodium morrhuate, P2G, and needlestick control were also investigated after 24 h (n = 4). In general, inflammation (CD43+, ED1+, and ED2+ cells) increased after prolotherapy injection compared to no-injection control but did not increase consistently compared to saline and needlestick control injections. This response varied by both location and proliferant. Inflammation was observed at 6 and 24 h postinjection but was resolved by 72 h compared to no-injection controls (p < 0.05). CD43+ leukocytes and ED2+ macrophages increased compared to needlestick and saline-injection control, respectively, 24 h postinjection (p < 0.05). Prolotherapy injections created an inflammatory response, but this response was variable and overall, not uniformly different from that caused by saline injections or needlestick procedures.

A systematic review of prolotherapy for chronic musculoskeletal pain.

OBJECTIVE: Prolotherapy, an injection-based treatment of chronic musculoskeletal pain, has grown in popularity and has received significant recent attention. The objective of this review is to determine the effectiveness of prolotherapy for treatment of chronic musculoskeletal pain. DATA SOURCES: We searched Medline, PreMedline, Embase, CINAHL, and Allied and Complementary Medicine with search strategies using all current and historical names for prolotherapy and injectants. Reference sections of included articles were scanned, and content area specialists were consulted. STUDY SELECTION: All published studies involving human subjects and assessing prolotherapy were included. MAIN RESULTS: Data from 34 case reports and case series and 2 nonrandomized controlled trials suggest prolotherapy is efficacious for many musculoskeletal conditions. However, results from 6 randomized controlled trials (RCTs) are conflicting. Two RCTs on osteoarthritis reported decreased pain, increased range of motion, and increased patellofemoral cartilage thickness after prolotherapy. Two RCTs on low back pain reported significant improvements in pain and disability compared with control subjects, whereas 2 did not. All studies had significant methodological limitations. CONCLUSIONS: There are limited high-quality data supporting the use of prolotherapy in the treatment of musculoskeletal pain or sport-related soft tissue injuries. Positive results compared with controls have been reported in nonrandomized and randomized controlled trials. Further investigation with high-quality randomized controlled trials with noninjection control arms in studies specific to sport-related and musculoskeletal conditions is necessary to determine the efficacy of prolotherapy.