Sex Dimorphism in Muscle Damage-Induced Inflammation

Evidence suggests that estrogen can provide a protective effect against muscle damage-induced inflammation. However, to date, no study has directly compared the muscle damage-induced intramuscular cytokines gene expression between men and women. Purpose: The purpose was to determine the intramuscular cytokine response to a bout of unaccustomed eccentric exercise in men and women. Methods: Untrained men (n=8, 22 ± 3y) and women (n=8, 20 ± 1y) completed a session of 80 unilateral maximal eccentric knee extensions. Vastus lateralis samples were collected and analyzed for gene expression of Interleukin (IL)-6, IL-10, IL-15, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-ß before exercise (BL), and 12 (12h) and 24 hours (24h) after exercise. Data were Results: A significant (p\u3c0.05) time x gender effect was found for IL-10 and TNF-α expression. IL-10 was increased at 12h (13.64 ± 4.22-fold) and 24h (29.34 ± 8.42-fold) compared to at BL for men, but there was no change for women. At 24h, IL-10 was greater for men than for women. Additionally, TNF-α was increased at 24h (7.78 ± 2.17-fold) compared to 12h (3.64 ± 1.36-fold) for men; no change was found for women. A significant time effect was found for IL-6 with an increased at 12h (3.23 ± 0.7-fold) and 24h (4.80 ± 1.57-fold) compared to BL. No changes were observed for IL-15 and TGF-ß expressions. Conclusion: In response to exercise-induced muscle damage, TNF-α and IL-10 gene expression increased in men but not in women. These results suggest that there is a sex dimorphic response in muscle damage-induced intramuscular pro-inflammatory and anti-inflammatory cytokines

Curcumin Mitigates Neuropathic-Induce Muscle Atrophy by Suppressing CaMK2/NF-kB Pathway

Neuropathy can induce inflammation that results in muscle atrophy. Curcumin has been shown to exert an anti-inflammatory effect, preserving muscle mass in diabetic rats. However, the mechanism of curcumin to preserve muscle mass in neuropathy is not known. PURPOSE: To examine the effect of curcumin on the intramuscular inflammation and muscle cross-sectional area (CSA) in a neuropathy rodent model. METHODS: Twelve rats were randomly assigned to three groups: sham (CON), spinal nerve ligation (SNL), and SNL+100curcumin/kg BW (100CUR). The right (R) lumbosacral section (i.e., L5/L6) of the spinal cord was ligated (SNL and 100CUR) or sham surgery (CON) was performed, whereas the contralateral side (left: L) was served as their own control. Rats were fed with a control diet without (i.e., CON and SNL) or with (i.e., 100CUR) curcumin supplementation for 4 weeks. Plantaris (left and right) and tibialis anterior (TA; right) muscles were collected. TA was stained for dystrophin to measure CSA. Left and right plantaris were analyzed for protein content for AChR, CaMK2, CaMK2Thr286, CaMK2Thr286/CaMK2, NF-κB, NF-κBSer536, NF-κBSer536/NF-κB, IL-1β, and GAPDH. Each protein was normalized to GAPDH then to the CON. RESULTS: A significant (p ≤ 0.05) group effect was observed for TA CSA and a group x leg interaction effect was observed for CaMK2Thr286/CaMK2, NF-κBSer536, IL-1β protein content. For muscle CSA, CON (9027.33 ± 603.39 μm2) and 100CUR (8853.68 ± 696.73 μm2) were larger than SNL (4771.01 ± 539.69 μm2). No difference was observed in CSA between CON and 100CUR. Additionally, when compared between left and right plantaris muscles, only SNL had greater CaMK2Thr286/CaMK2 (R: 2.63 ± 1.87 vs. L: 1.56 ± 1.65), NF-κBSer536 (R: 1.85 ± 0.83 vs. L: 0.55 ± 0.33), and IL-1β (R: 2.11 ± 1.32 vs. L: 0.65 ± 0.29) protein content in the right than the left leg, whereas, no difference was observed for left leg among groups. For NF-κBSer536, SNL (1.85 ± 0.83) was greater than 100CUR (0.91 ± 0.52) in the right leg. No significant differences were observed for AChR, CaMK2, CaMK2Thr286, and NF-κBSer536/NF-κB. CONCLUSION: In a neuropathic model, muscle atrophy was observed with concomitant increase in CaMK2/NF-κB/IL-1β activation in the ipsilateral plantaris. Curcumin supplementation appears to mitigate this inflammatory response and muscle mass loss

Ginger Root Extract Increases Mitochondrial Fission and Mitophagy in Diabetes Mellitus Rats

Diabetes (DM) is accompanied by mitochondrial dysfunction (i.e., mitochondria fission/fusion and mitophagy) in which result in an accumulation of damaged mitochondria and further impaired insulin resistance. Ginger root extract (GRE) has been shown to improve mitochondrial biogenesis and decreased respiratory coefficient in DM model, however, the effect of GRE on the basal mitochondria fission/fusion and mitophagy state is limited. PURPOSE: To determine the effect of GRE on mitochondria fission/fusion and mitophagy transcript abundance in rats with diabetes induced by high-fat diet (HFD) with streptozotocin (STZ). METHOD: Sprague-Dawley rats were randomly divided into 3 groups: standard diet (STD; n=11), HFD with 35 mg/kg of STZ (DM; n=11), and HFD+STZ with 0.75% w/w GRE (GRE; n=10). After 7 weeks, soleus samples were collected and analyzed for gene expression for fission/fusion (DRP, MFN) and mitophagy (PINK1, PARKIN, BECN1, LC3A, LC3B, P62). RESULT: A significant (p\u3c0.05) condition effect was found for PINK1, DRP, LC3A, LC3B, P62, and autophagic flux. For fission/fusion, GRE had significantly greater DRP (2.27±0.9-fold vs. 0.47±0.1-fold) than DM and no difference was found for MFN. For mitophagy, GRE had significantly greater PINK1 (1.59±0.55-fold vs. 0.31±0.06-fold), LC3A (1.81±0.65-fold vs. 0.13±0.02-fold), LC3B (2.71±0.92-fold vs. 0.66±0.25-fold), P62 (3.25±1.24-fold vs. 0.43±0.12-fold), and autophagy flux (4.5±1.06-fold vs. 2.41±0.36-fold) than DM and greater LC3B (2.71±0.92-fold vs. 1±0.06-fold), P62 (3.25±1.24-fold vs. 1±0.21-fold), and autophagic flux (4.5±1.06-fold vs. 1±0.26-fold) than STD. No difference was found for PARKIN and BECN1. CONCLUSION: In DM rats, GRE increased basal expression of mitochondria fission, degradation tag (PINK1), and autophagolysosome (LC3A, LC3B, P62, autophagic flux) markers, suggesting a potential increased in mitochondrial fission and mitophagy capacity

Geranylgeraniol Increases Autophagy and Mitophagy Gene Expression in Soleus of Rats with Diabetes

The autophagy and mitophagy (selective autophagy for mitochondria) processes are important in maintaining muscle homeostasis, e.g., removing damaged mitochondria. With diabetes, skeletal muscle autophagy decreases. Geranylgeraniol (GG) has been shown to reduce mitochondrial damage; however, the effect of GG on basal autophagy and mitophagy in diabetic rats is not known. PURPOSE: To determine the effect of GG on selective autophagy and mitophagy genes in rats with diabetes induced by a high-fat diet (HFD) with streptozotocin (STZ). METHODS: Sprague-Dawley rats were divided into three groups: normal diet (CON; n=11), HFD with 35 mg/kg body weight of STZ (HFD; n=9), and HFD/STZ with 800 mg/kg body weight of GG (GG; n=9). On the 7th week, soleus muscles were collected and analyzed for gene expression of LC3A, LC3B, P62, PINK1, PARKIN, DRP, and MFN. Gene data were normalized to CON. RESULTS: A significant (p \u3c 0.05) condition effect was found for autophagy (LC3A, LC3B, and P62) and mitophagy (PINK1, DRP, and MFN) gene expression. For autophagy, HFD (0.14 ± 0.03-fold) had significantly lower LC3A than CON (1.00 ± 0.22-fold), lower LC3B (0.67 ± 0.26-fold vs. 2.37 ± 0.72-fold) and P62 (0.44 ± 0.13-fold vs. 1.70 ± 0.35-fold) than GG. HFD trended to have lower LC3A than GG (0.14 ± 0.03-fold vs. 0.86 ± 0.26-fold; p = 0.066) while CON trended to have lower LC3B than GG (1.00 ± 0.07-fold vs. 2.37 ± 0.72-fold; p = 0.078). For mitophagy, HFD (0.32 ± 0.07-fold) and GG (0.51 ± 0.15-fold) had significantly lower PINK1 than CON (1.00 ± 0.13-fold). Further, HFD had lower MFN than GG (0.31 ± 0.08-fold vs. 1.46 ± 0.25-fold) and lower DRP than CON (0.48 ± 0.11-fold vs. 1.00 ± 0.15-fold). HFD trended to have lower PARKIN than GG (0.46 ± 0.08-fold vs. 1.38 ± 0.38-fold; p = 0.053) with no difference between GG and CON. CONCLUSION: In comparison to HFD, GG consumption improved the basal transcript abundance of the selective skeletal muscle autophagic and mitophagic genes, which could indicate an increased capacity to remove damaged mitochondria in diabetic rats

Geranylgeraniol Supplementation Mitigates Muscle Atrophy with Mitochondrial Quality Improvement in Diabetic Rats

With diabetes, skeletal muscle mitochondrial quality control (mitochondrial fusion, fission & macro-autophagy) is impaired. Geranylgeraniol (GG) is shown to have a protective effect on preventing mitochondrial damage and muscle health; however, the effect of GG on a diabetic model is not known. PURPOSE: To determine the effect of GG on mitochondrial quality control and muscle cross-sectional area (CSA) in diabetic rats. METHODS: Thirty-five Sprague-Dawley rats were divided into three diet groups: control diet (CON), high-fat diet with 35 mg/kg body weight of streptozotocin (HFD), and HFD with 800 mg/kg body weight of GG (GG). Due to the limited sample, a total of 21 (CON: n = 7; HFD: n = 7; GG: n = 7) rats’ muscle samples were used for this report. The soleus muscles were harvested after 7-weeks of feeding and were analyzed for OPA1, MFN2, DRP1, pDRP, PINK1, Parkin, LC3A, and LC3B protein content using western blot analysis. Muscle CSA were assessed using Image J. RESULTS: A significant (p \u3c 0.05) condition effect was observed for MFN2, DRP, LC3A, and LC3B protein contents and muscle CSA. For mitochondrial fusion, GG (0.21 ± 0.08) had lower MFN2 than CON (0.43 ± 0.04; p = 0.007) and HFD (0.65 ± 0.08; p = 0.010). For mitochondrial fission, GG (0.26 ± 0.07) had lower DRP than HFD (0.59 ± 0.07; p = 0.019). For macro-autophagy, GG (1.08 ± 0.28) had lower LC3A than CON (2.81 ± 0.55; p = 0.028) and HFD (3.99 ± 0.57; p = 0.010); whereas GG (0.63 ± 0.21) had lower LC3B than HFD (1.93 ± 0.24; p = 0.012). No significant differences were observed for OPA1, pDRP, PINK1, Parkin, and LC3B/A. For muscle size, CON (10,092.88 ± 104.67µm2) had larger CSA than GG (7284.69 ± 70.91µm2, p = 0.001) and HFD (5615.59 ± 59.97µm2; p = 0.001), whereas GG (7284.69 ± 70.91µm2) had larger CSA than HFD (5615.59 ± 59.97µm2; p = 0.001). CONCLUSION: GG supplementation could prevent mitochondrial fragmentation (reduction in DRP), thus, potentially resulting in a decreased demand for mitochondrial fusion (reduction in MFN2). In addition, a greater rate of autophagosome degradation than formation (reduction in LC3A and LC3B) was observed (indicative of an increase in macro-autophagy). Improvement in mitochondrial quality could potentially contribute to attenuating the reduction of muscle size in diabetic rats with GG supplementation

The Effect of Geranylgeraniol on Satellite Cells Myogenic State in Type 2 Diabetic Rats

Type 2 Diabetes (T2D) is associated with chronic inflammation, which can contribute to impaired satellite cells (SC) myogenic state that may result in muscle atrophy. Geranylgeraniol (GGOH) has shown to prevent muscle atrophy, reduce inflammatory markers, and increase SC content; however, the effect of GGOH on SC myogenic state in T2D rats is not known. PURPOSE: To examine the effects of GGOH on SC myogenic state and muscle cross-sectional area (CSA) in T2D rats. METHODS: 21 Sprague-Dawley rats were fed a control diet (CON; n=7), a high-fat diet with 35 mg/kg of streptozotocin (HFD; n=7), and HFD with 800mg/kg body weight of GGOH (GG; n=7). In the 8th week, the right soleus muscle was analyzed for protein expression for Pax7, MyoD, myostatin, and GAPDH, and protein content was normalized to GAPDH. The left soleus muscle was co-stained with Pax7, MyoD, and myostatin using immunohistochemistry and analyzed for muscle CSA. Counted SC were normalized to 100 fibers. RESULTS: A significant (p \u3c 0.05) condition effect was observed for MyoD and myostatin protein expression. For MyoD, HFD (1.41 ± 0.09 A.U.) was lower than CON (2.24 ± 0.21 A.U.) and GG (2.62 ± 0.43 A.U.). For myostatin, HFD (0.42 ± 0.06 A.U.) was lower than CON (0.91 ± 0.09 A.U.). Additionally, a significant condition effect was observed for the number of cells that presented Pax7+/MyoD- and Pax7+/myostatin+. For Pax7+/MyoD-, HFD (0.039 ± 0.004) and GG (0.035 ± 0.004) had lower cell counts than CON (0.064 ± 0.010). For Pax7+/myostatin+, HFD (0.034 ± 0.003) had lower cell counts than GG (0.065 ± 0.010) and CON (0.057 ± 0.004). A significant condition effect was observed for CSA where CON (7099.89 ± 187.33 μm2) was larger than HFD (4351.02 ± 127.46 μm2) and GG (5584.61 ± 208.01 μm2), while GG (5584.61 ± 208.01 μm2) was larger than HFD (4351.02 ± 127.46 μm2). CONCLUSION: GGOH supplementation to T2D rats mitigated muscle mass loss (increased MyoD expression with no change in MyoD+ SC). Despite no differences in SC myogenic state (proliferative and differentiation) among groups, GGOH appeared to mitigate the reduction in the quiescent SC pool (Pax7+/myostatin+) observed in HFD. Given the importance of quiescent SC pool on retaining myogenic potential, which is essential for muscle hypertrophy and regeneration, supplementing GGOH to T2D rats could improve muscle health

Sex Differences in The Accuracy of WUT (Weight, Urine Color, Thirst) Diagrams Assessing Hydration Status

The WUT (Weight, Urine Color, Thirst) Venn diagram is a practical method to assess hydration status using percent body mass loss (%BML), urine color (UCOL), and thirst perception (TP). However, sex differences and the accuracy of WUT diagrams between males and females has not yet been investigated. PURPOSE: To observe sex differences in the accuracy of WUT diagrams assessing hydration status. METHODS: 8 males [M] (age: 21 ± 3; mass: 76.3 ± 15.6 kg) and 5 females [F] (age: 22 ± 2; mass: 60.5 ± 13.6) visited the laboratory twice a day (morning (7:00am-9:00am) and afternoon (2:00pm-4:00pm)) for six days as free-living for the first three consecutive days and euhydrated (urine specific gravity (USG) \u3c 1.020) for the last three consecutive days. During each visit, TP, body mass (BM), USG, UOSM, UCOL, and plasma osmolality (POSM) were collected. Values of USG \u3e1.020, UOSM \u3e700, and POSM \u3e290 indicated dehydration status. TP \u3e5, UCOL \u3e5, and %BML \u3e1% values were used as dehydration thresholds for WUT scores. Total WUT score (0-3) was determined by the total amount of respective dehydration markers identified. One-way ANOVA was used to analyze differences in POSM, UOSM, and USG between the different WUT scores for both sexes. Receiver operating characteristics analysis was used to calculate sensitivity (SENS) and specificity (SPEC) identifying dehydration or euhydration with WUT scores. RESULTS: For POSM, WUT3 (M: 291 ± 5; F: 286 ± 0 mOsmol), WUT2 (289 ± 6; 286 ± 7), WUT1 (286 ± 5; 286 ± 6), and WUT0 (289 ± 5; 285 ± 7) were not different between sexes (p \u3e .05). For USG, WUT3 (1.022 ± .004; 1.020 ± .000), WUT2 (1.019 ± .008; 1.020 ± .007), WUT1 (1.015 ± .006; 1.010 ± .005), and WUT0 (1.010 ± .006; 1.008 ± .006) were not different between sexes (p \u3e .05). For UOSM, WUT3 (819 ± 147; 744 ± .000 mOsmol), WUT2 (679 ± 244; 788 ± 261), WUT1 (521 ± 266; 461 ± 212), and WUT0 (383 ± 212; 322 ± 203) were not different between sexes (p \u3e .05). For POSM, WUT2SPEC was higher in M (WUT2Mspec, .860) than F (WUT2Fsepc, .786) while WUT3, WUT1, and WUT0 were similar between sexes (WUT3Mspec, .965; WUT3Fspec, .976; WUT1Mspec, .526; WUT1Fspec, .380). For USG, WUT2SENS was higher in F (WUT2Fsens, .889) than M (WUT2Msens, .571) while WUT3, WUT1, and WUT0 were similar between sexes (WUT3Msens, .238; WUT3Fsens, .111; WUT1Msens, .905; WUT1Fsens, .889). For UOSM, SPEC and SENS were similar between sexes for each WUT score. CONCLUSION: There are no sex differences in POSM, USG, and UOSM between WUT0-WUT3. However, based on SPEC and SENS, WUT3 and WUT0 can accurately detect hydration status in both sexes. WUT2 might be used to detect hydration status only for females

Habitual Fluid Intake Does Not Affect Sleep Parameters in Young Women.

Sleep is essential for optimal physical performance, cognitive function, recovery, and overall health. Similarly, hydration status has been shown to influence physical and cognitive functions. Dehydration can lead to impaired cognition and possibly impaired sleep. However, the effect of habitual total water intake (TWI) on sleep and recovery measures have not been examined. PURPOSE: To examine the effect of TWI on sleep and recovery measures in young women. METHODS: Twenty-two young women (age: 22.8±4.6 yrs; body mass: 60.0±9.5 kg; height: 164.6±6.1 cm) collected fluid intake and food consumption information across a 5 day period, and average TWI was calculated based on them. Then, participants were categorized in either High Drinker (HD; ³ 2.5L/day; n=13; age: 23.0±3.6 yrs) or Low Drinker (LD; £ 1.6 L/day; n=9; age: 23±3yrs). Sleep and recovery measures were collected using a wearable sleep-tracking device that participants wore for 5 consecutive days. Sleep and recovery measures were compared between HD and LD, using a two-tailed independent t-test and effect sizes (ES). ES were identified as either small (0.2-0.49), medium (0.5-0.79), and large (\u3e0.8). RESULTS: No significant differences were found in resting heart rate (HD: 63.7±4.6 bpm, LD: 61.9± 5.2 bpm; p=0.40), heart rate variability (HD: 58.2±14.1 ms, 69.6±40.9 ms; p=0.44), slow wave sleep (SWS) (HD: 1.5±0.4 hrs, LD: 1.4±0.3 hrs; p=0.55), SWS percentage (HD: 19.3±3.8 %, LD: 18.7±3.7 %; p=0.17) sleep consistency (HD: 65.2±15.6 %, LD: 63.3±7. 3%; p=0.71), and sleep efficiency (HD: 90.5±3.1 %, LD: 90.4±2.0 %; p=0.91). While there were no statistical differences, rapid eye movement (REM) sleep (HD: 2.0±0.4 hrs, LD: 1.6±0.7 hrs; p=0.17) and REM percentage (HD: 26.1±3.8 %, LD: 21.5±7.8 %; p=0.13) displayed the largest differences, with REM indicating a medium ES (d=0.70) and REM percentage having a large ES (d=0.80). CONCLUSION: Habitual fluid intake might not impact sleep measurement. However, based on ES, REM sleep and REM percentage potentially display a trend. Still, more research is necessary to further determine any correlations

Sleep Duration is Increased Following Muscle Damaging Exercise in Hot Environmental Conditions

Sleep and recovery measures are typically negatively affected by a muscle-damaging bout of exercise. However, it remains unknown if the additive effects of hot environmental conditions, resulting in increased core temperature and other thermoregulatory responses during the exercise bout, further progress changes in quantity and performance quality of sleep duration. PURPOSE: To investigate the effect of muscle-damaging exercise in the heat, compared to a thermoneutral condition, on sleep and recovery measures. METHODS: Ten healthy males (age: 23 ± 3yr; body mass: 78.7 ± 11.5kg; height: 176.9 ± 5cm; lactate threshold [LT]: 9.7 ± 1.0km.hr-1) performed two protocols in a randomized, counterbalanced order of downhill running (DHR) for 30-minutes at the LT in either a thermoneutral (ambient temperate [Tamb], 20°C; relative humidity [RH], 20%) or hot environmental condition (Tamb, 35°C; RH, 40%) at a -10% gradient. Sleep and recovery measures were collected from a wearable sleep device participants wore the night after the DHR. Differences in sleep and recovery measures following DHR in the heat compared to a thermoneutral condition were analyzed using paired samples T-tests. RESULTS: Sleep hours, restorative sleep hours, rapid eye movement (REM) sleep hours, and slow wave sleep (SWS) hours were all greater following the heat condition (mean ± SD; sleep hours: 6.70 ± 0.74hr, p = 0.040; restorative sleep hours: 3.31 ± 0.90hr, p = 0.012; REM sleep hours: 1.70 ± 0.64hr, p = 0.046; SWS hours: 1.61 ± 0.35hr, p = 0.015) compared to the thermoneutral condition (sleep hours: 5.24 ± 1.75hr; restorative sleep hours: 2.45 ± 1.11hr; REM sleep hours: 1.23 ± 0.68hr; SWS: 1.22 ± 0.53hr). Also, recovery was higher following the heat condition (recovery: 75.88 ± 15.31, p = 0.023) compared to the thermoneutral condition (recovery: 50.75 ± 21.46). Sleep efficiency, sleep disturbance, sleep deprivation, sleep score, %REM, %SWS, light sleep, resting heart rate, and heart rate variability were not different between conditions (ps \u3e 0.05). CONCLUSION: Following muscle-damaging exercise in the heat, sleep and recovery duration measures were increased compared to a thermoneutral condition. These findings suggest that performing muscle-damaging exercises in hot conditions may require a greater amount of sleep for optimal recovery

Relationships between Morning and Afternoon WUT (Weight, Urine Color, and Thirst) Criteria and Hydration Markers

A Venn diagram decision tool consisting of weight, urine color, and thirst (WUT) is suggested to measure hydration status. The WUT Venn diagram has been used as a practical hydration status assessment tool; however, this relationship has only been investigated using a first-morning urine sample. PURPOSE: To investigate relationships between morning and afternoon WUT criteria, blood and urine markers. METHODS: Eight men (age: 21 ± 3; mass: 76.3 ± 15.6 kg) and five women (age: 22 ± 2; mass: 60.5 ± 13.6 kg) completed the study. Body mass, urine color, urine specific gravity (USG), urine osmolality (UOSM), thirst level, and plasma osmolality (POSM) were collected as a first-morning and afternoon spot urine (2:00-4:00 CST) for 3 consecutive days in a free-living situation and 3 consecutive days in a euhydrated state. Body mass loss \u3e1%, urine color \u3e5, and thirst level ≥5 were used as dehydration thresholds. The number of markers that indicated dehydration levels were counted and categorized into either 3, 2, 1, or 0 WUT markers indicating dehydration (defined by either USG, UOSM, or POSM). One-way ANOVA with Tukey pairwise comparisons were used to assess differences in USG, UOSM, and POSM between different numbers of WUT markers. Receiver operating characteristics analysis was performed to calculate the predictive value of 0, 1, 2, or 3 hydration markers in detecting a dehydrated or euhydrated state. RESULTS: Morning and afternoon 1, 2, and 3 WUT markers were not significantly different (ps \u3e .05) for USG and POSM. Morning and afternoon 0, 2, and 3 WUT markers were not significantly different for UOSM. Morning and afternoon 3 WUT resulted in a specificity of 0.984 and 1.000, 0.984 and 1.000, and 0.956 and 0.981 for USG \u3e 1.020, UOSM \u3e 700mOsm, and POSM \u3e 290mOsm, respectively. Meeting at 2 WUT for morning and afternoon resulted in a specificity of 0.820 and 0.985, and 0.806 and 0.984 for USG and UOSM, respectively. Meeting at 1 WUT for morning and afternoon resulted in a sensitivity of 1.000 and 0.813 for UOSM. CONCLUSION: These results suggest that when 2 or 3 WUT markers are met, urine and blood hydration markers indicate dehydration, and when 1 WUT marker is met, UOSM indicates not dehydrated. The WUT Venn diagram can assess hydration status when an afternoon spot urine sample is used