Methodological aspects of in vivo axial loading in rodents: a systematic review.

Axial loading in rodents provides a controlled setting for mechanical loading, because load and subsequent strain, frequency, number of cycles and rest insertion between cycles, are precisely defined. These methodological aspects as well as factors, such as ovariectomy, aging, and disuse may affect the outcome of the loading test, including bone mass, structure, and bone mineral density. This review aims to overview methodological aspects and modifying factors in axial loading on bone outcomes. A systematic literature search was performed in bibliographic databases until December 2021, which resulted in 2183 articles. A total of 144 articles were selected for this review: 23 rat studies, 74 mouse studies, and 47 knock out (KO) mouse studies. Results indicated that peak load, frequency, and number of loading cycles mainly affected the outcomes of bone mass, structure, and density in both rat and mouse studies. It is crucial to consider methodological parameters and modifying factors such as age, sex-steroid deficiency, and disuse in loading protocols for the prediction of loading-related bone outcomes.

Mechanical loading modulates phosphate related genes in rat bone.

Mechanical loading determines bone mass and bone structure, which involves many biochemical signal molecules. Of these molecules, Mepe and Fgf23 are involved in bone mineralization and phosphate homeostasis. Thus, we aimed to explore whether mechanical loading of bone affects factors of phosphate homeostasis. We studied the effect of mechanical loading of bone on the expression of Fgf23, Mepe, Dmp1, Phex, Cyp27b1, and Vdr. Twelve-week old female rats received a 4-point bending load on the right tibia, whereas control rats were not loaded. RT-qPCR was performed on tibia mRNA at 4, 5, 6, 7 or 8 hours after mechanical loading for detection of Mepe, Dmp1, Fgf23, Phex, Cyp27b1, and Vdr. Immunohistochemistry was performed to visualise FGF23 protein in tibiae. Serum FGF23, phosphate and calcium levels were measured in all rats. Four-point bending resulted in a reduction of tibia Fgf23 gene expression by 64% (p = 0.002) and a reduction of serum FGF23 by 30% (p<0.001), six hours after loading. Eight hours after loading, Dmp1 and Mepe gene expression increased by 151% (p = 0.007) and 100% (p = 0.007). Mechanical loading did not change Phex, Cyp27b1, and Vdr gene expression at any time-point. We conclude that mechanical loading appears to provoke both a paracrine as well as an endocrine response in bone by modulating factors that regulate bone mineralization and phosphate homeostasis.

Mechanical stress regulates bone regulatory gene expression independent of estrogen and vitamin D deficiency in rats.

Mechanical stress determines bone mass and structure. It is not known whether mechanical loading affects expression of bone regulatory genes in a combined deficiency of estrogen and vitamin D. We studied the effect of mechanical loading on the messenger RNA (mRNA) expression of bone regulatory genes during vitamin D and/or estrogen deficiency. We performed a single bout in vivo axial loading with 14 N peak load, 2 Hz frequency and 360 cycles in right ulnae of nineteen weeks old female control Wistar rats with or without ovariectomy (OVX), vitamin D deficiency and the combination of OVX and vitamin D deficiency (N = 10/group). Total bone RNA was isolated 6 hours after loading, and mRNA expression was detected of Mepe, Fgf23, Dmp1, Phex, Sost, Col1a1, Cyp27b1, Vdr, and Esr1. Serum levels of 25(OH)D, 1,25(OH)2 D and estradiol were also measured at this time point. The effect of loading, vitamin D and estrogen deficiency and their interaction on bone gene expression was tested using a mixed effect model analysis. Mechanical loading significantly increased the mRNA expression of Mepe, and Sost, whereas it decreased the mRNA expression of Fgf23 and Esr1. Mechanical loading showed a significant interaction with vitamin D deficiency with regard to mRNA expression of Vdr and Esr1. Mechanical loading affected gene expression of Mepe, Fgf23, Sost, and Esr1 independently of vitamin D or estrogen, indicating that mechanical loading may affect bone turnover even during vitamin D deficiency and after menopause.

Dried Blood Spot Thyroglobulin as a Biomarker of Iodine Status in Pregnant Women.

Context: Thyroglobulin (Tg) could be a sensitive biomarker of iodine nutrition in pregnant women (PW). A dried blood spot (DBS) assay would simplify collection and transport in field studies. Objectives: Our aims were to (1) establish and test a reference range for DBS-Tg in PW; (2) determine whether co-measurement of Tg antibodies (Abs) is necessary to define population iodine status. Design, Setting, and Participants: Standardized cross-sectional studies of 3870 PW from 11 countries. For the DBS-Tg reference range, we included TgAb-negative PW (n = 599) from 3 countries with sufficient iodine intake. Main Outcome Measures: We measured the urinary iodine concentration and DBS thyroid-stimulating hormone, total thyroxin, Tg, and TgAb. Results: In the reference population, the median DBS-Tg was 9.2 µg/L (95% confidence interval, 8.7 to 9.8 µg/L) and was not significantly different among trimesters. The reference range was 0.3 to 43.5 µg/L. Over a range of iodine intake, the Tg concentrations were U-shaped. Within countries, the median DBS-Tg and the presence of elevated DBS-Tg did not differ significantly between all PW and PW who were TgAb-negative. Conclusions: A median DBS-Tg of ∼10 µg/L with <3% of values ≥44 µg/L indicated population iodine sufficiency. Concurrent measurement of TgAb did not appear necessary to assess the population iodine status.

Anemia, Iron Deficiency and Iodine Deficiency among Nepalese School Children.

OBJECTIVES: To assess iodine and iron nutritional status among Nepalese school children. METHODS: A cross-sectional, community based study was conducted in the two districts, Ilam (hilly region) and Udayapur (plain region) of eastern Nepal. A total of 759 school children aged 6-13 y from different schools within the study areas were randomly enrolled. A total of 759 urine samples and 316 blood samples were collected. Blood hemoglobin level, serum iron, total iron binding capacity and urinary iodine concentration was measured. Percentage of transferrin saturation was calculated using serum iron and total iron binding capacity values. RESULTS: The mean level of hemoglobin, serum iron, total iron binding capacity, transferrin saturation and median urinary iodine excretion were 12.29 ± 1.85 g/dl, 70.45 ± 34.46 µg/dl, 386.48 ± 62.48 µg/dl, 19.94 ± 12.07 % and 274.67 µg/L respectively. Anemia, iron deficiency and iodine deficiency (urinary iodine excretion <100 µg/L) were present in 34.5 %, 43.4 % and 12.6 % children respectively. Insufficient urinary iodine excretion (urinary iodine excretion <100 µg/L) was common in anemic and iron deficient children. CONCLUSIONS: Iron deficiency and anemia are common in Nepalese children, whereas, iodine nutrition is more than adequate. Low urinary iodine excretion was common in iron deficiency and anemia.

Urinary iodine excretion and thyroid function status in school age children of hilly and plain regions of Eastern Nepal.

BACKGROUND: Iodine deficiency is a major public health problem in many developing countries including Nepal. The present study was designed to investigate the urinary iodine excretion (UIE), thyroid function status and household salt iodine content (SIC) in school-aged children (SAC) and to establish the relationships between these factors. METHODS: A community-based cross sectional study was conducted in selected schools of two districts, Tehrathum and Morang, lying in the hill and plain region of eastern Nepal respectively. A total of 640 SAC, (Tehrathum n = 274 and Morang n = 366) aged 6-11 years, were assessed for UIE and household SIC. Among the 640 children, 155 consented to blood samples (Tehrathum n = 78 and Morang n = 77) to test for serum thyroglobulin (Tg), thyroid stimulating hormone (TSH), free triiodothyronine (fT3) and free thyroxine (fT4). UIE was measured by ammonium persulfate digestion method. SIC was measured by iodometric titration method and Tg, TSH, fT4 and fT3 were measured by immunoassay based kit method. RESULTS: In Tehrathum and Morang, 9.5 and 7.7% of SAC had UIE values of UIE <100 µg/L while 59.5 and 41% had iodine nutrition values of >299 µg/L, with median UIE of 345.65 and 270.36 µg/L respectively. The overall medians were as follows, Tg 14.29 µg/L, fT3 3.94 pmol/L, fT4 16.25 pmol/L and TSH 3.61 mIU/L. There was a negative correlation between UIE and Tg (r = -0.236, p = 0.003) and a positive correlation between UIE and SIC (r = 0.349, p < 0.0001). We found 19.5%, n = 15 and 16.7%, n = 13 subclinical hypothyroid cases in Morang and Tehrathum respectively. Iodometric titration showed only 6.4% (n = 41) of the samples had household SIC <15 ppm. Multivariate analysis revealed that use of packaged salt by SAC of Tehrathum district correlated with higher UIE values. CONCLUSIONS: Our focused data suggests that collaborative universal salt iodization (USI) programs are improving the health of children in the Tehrathum and Morang districts of Nepal. We also found that excessive iodine in a large portion of the study groups is a substantial concern and iodine intervention programs need to deal with both deficient and excessive iodine scenarios that can both be present simultaneously in study populations.

Subclinical Hypothyroidism and Elevated Thyroglobulin in Infants with Chronic Excess Iodine Intake.

BACKGROUND: Acute iodine excess in newborns can cause hypothyroidism, but there are limited data on the effects of iodine excess on thyroid function in older infants. The aim of this study was to measure the effects of chronic excess iodine intake on thyroid function in 6-24-month-old infants. METHODS: In this cross-sectional study, infants (n=696) in eastern Nepal were studied. Spot urine samples, venous blood samples, and household salt samples were collected, and urinary iodine concentration (UIC), serum free thyroxine (fT4), thyrotropin (TSH), thyroglobulin (Tg), and titrated household salt iodine concentration (SIC) were measured. Daily iodine intake was calculated from UIC based on estimates of urine volume at this age. RESULTS: Median (25th-75th percentile) household SIC was 89 (70-149) ppm, while national legislation stipulates a fortification level of 50 ppm. Median UIC was 407 (312-491) µg/L; 76% of infants had a UIC >300 µg/L, suggesting iodine excess. Calculated mean iodine intake in 12-24-month-old infants was 220 µg/day, exceeding the recommended safe upper limit for iodine at this age (200 µg/day). Among the infants, 15.8% had an elevated Tg, 7.4% had subclinical hypothyroidism, but <1% had overt hypothyroidism. UIC was not a significant predictor of thyroid function, thyroid hormones, or Tg. CONCLUSION: In 6-24-month-old infants exposed to excessive iodine intake, ∼7% have subclinical hypothyroidism but <1% have overt hypothyroidism. These findings suggest the thyroid in late infancy is already able to adapt to high iodine intakes and, in most cases, maintain euthyroidism.

Plasma zinc levels, anthropometric and socio-demographic characteristics of school children in eastern Nepal.

BACKGROUND: Zinc deficiency is a major public health problem in many developing countries including Nepal. The present study was designed to assess the prevalence of zinc deficiency and to study the association of zinc deficiency with anthropometric and socio-demographic variables, in school children of eastern Nepal. METHODS: This cross-sectional study included total 125 school children of age group 6-12 years from Sunsari and Dhankuta districts of eastern Nepal. Plasma zinc level was estimated by Flame Atomic Absorption Spectroscopy. RESULTS: The Median interquartile range (IQR) values of zinc in the two districts Sunsari and Dhankuta were 5.9 (4.4, 7.9) µmol/L and 5.8 (4.3, 8.4) µmol/L respectively. A total of 55 children (87.3%) in Sunsari and 52 (83.9%) in Dhankuta had zinc deficiency, no significant difference was observed in the Median (IQR) plasma zinc levels (p = 0.9) and zinc deficiency patterns (p = 0.3) of the two districts. Significant differences were observed in the plasma zinc levels (p = 0.02) and zinc deficiency patterns (p = 0.001), of the school children having age groups 6-8 years than in 9-10 and 11-12 years of age, and zinc deficiency patterns between male and female school children (p = 0.04) respectively. CONCLUSIONS: The present study showed higher prevalence of zinc deficiency among school children in eastern Nepal. In our study, zinc deficiency was associated with both sex and age. The findings from the present study will help to populate data for policy implementation regarding consumption and supplementation of zinc.

Household salt iodine content estimation with the use of rapid test kits and iodometric titration methods.

BACKGROUND: Universal salt iodization remains the best strategy for controlling iodine deficiency disorders in Nepal. AIMS: This study was designed to study the salt types and the household salt iodine content of school aged children in the hilly and the plain districts of eastern Nepal. MATERIAL AND METHODS: This cross-sectional study was carried out on school children of seven randomly chosen schools from four districts, namely, Sunsari, Dhankuta, Sankhuwasabha and Tehrathum of eastern Nepal. The school children were requested to bring two teaspoonfuls (approx. 12-15 g) of the salt which was consumed in their households, in a tightly sealed plastic pouch. The salt types were categorized, and the salt iodine content was estimated by using rapid test kits and iodometric titrations. The association of the salt iodine content of the different districts were tested by using the Chi-square test. The sensitivity, specificity, positive predictive values, and negative predictive values of the rapid test kits were compared with the iodometric titrations. RESULTS: Our study showed that mean±SD values of the salt iodine content in the four districts, namely, Sunsari, Dhankuta, Sankhuwasabha and Tehrathum were 34.2±17.9, 33.2±14.5, 27.4±15.1 and 48.4±15.6 parts per million (ppm). There were 270 (38.2%) households which consumed crystal salt and 437(61.8%) of the households consumed packet salts. CONCLUSIONS: Our study recommends a regular monitoring of the salt iodization programs in these regions. More families should be made aware of the need to ensure that each individual consumes iodized salt.

Iodine status after iodized salt supplementation in schoolchildren of eastern Nepal.

This study was designed to assess the urinary iodine concentrations of schoolchildren at baseline and after iodized salt supplementation in eastern region of Nepal. A cross sectional study was conducted from August 2009 to July 2011 among schoolchildren of three eastern districts of Nepal: Sunsari, Dhankuta, and Tehrathum. A sample of 828 school age children from the three districts was chosen for the study after obtaining written consent from their guardians. The schoolchildren treatment group (n=300) was provided with a supplement of iodized salt for six months. Urinary iodine concentration was estimated by ammonium persulfate digestion microplate method at baseline and after supplementation. Urinary iodine controls L1, L2 (Seronorm, Norway) were analyzed to obtain intra-assay CVs (L1 = 7.4%, L2 = 3.3%) and inter assay CVs (L1=23.5%, L2=11.26%). Median interquartile range urinary iodine concentration in the three districts: Sunsari, Dhankuta and Tehrathum at baseline versus intervention were 272.0 (131.5-473.0) microg/l versus 294.0 (265.0-304.0) microg/l (p=0.379), 247.0 (144.5-332.32) versus 361.0 (225.66-456.52) microg/l (p<0.001), and 349.5 (203.75-458.09) microg/l versus 268.76 (165.30-331.67) microg/l (p<0.001), respectively. This study indicated improved iodine status and increased median urinary iodine concentration after iodized salt supplementation. Regular monitoring of population urinary iodine concentration at national and regional levels should be performed to ensure that all individuals have optimal delivery of iodine nutrition.

Non-enzymatic antioxidant status and biochemical parameters in the consumers of Pan Masala containing tobacco.

BACKGROUND: Tobacco consumption is one of the leading causes of oral submucous fibrosis, oral cancer and even premature death. The present study was designed to compare the biochemical parameters and non- enzymatic antioxidant status and the lipid peroxidation products in pan masala tobacco users as compared with age-matched non-user controls. METHODS: Pan masala and tobacco users of age 33.2±9.94 years and age-matched controls (31.2±4.73 years) were enrolled for the study. Plasma levels of vitamin E, vitamin C, albumin, bilirubin, uric acid, glucose, urea, creatinine, aspartate amino transferase (AST), alanine amino transferase (ALT) were measured by standard methods. Serum malondialdehyde (MDA) levels were estimated as a measure of lipid peroxidation. RESULTS: In the pan masala tobacco users, as compared to the controls, the level of vitamin C (68.5±5.9 vs 97.9±9.03 µmol/L, p≤0.05) vitamin E (18.4±5.3 vs 97.9±9.03 µmol/L, p≤0.001), albumin (37.5±7.01 vs 44.3±9.99 g/L, p≤0.001), and malondialdehyde (10.8±1.29 vs 1.72±1.15 nmol/ml, p≤0.001 ) were found to be significantly altered. Malondialdehyde was significantly correlated with vitamin E (r=1.00, p<0.001) and vitamin C (r =1.00, p<0.001) in pan masala tobacco users. Serum levels of AST (31.0±16.77 IU) and ALT (36.7±31.3 IU) in the pan masala tobacco users were significantly raised as compared to the controls (AST, 25.2±9.51 IU, p=0.038; ALT, 26.2±17.9 IU, p=0.038). CONCLUSION: These findings suggest that pan masala tobacco users are in a state of oxidative stress promoting cellular damage. Non-enzymatic antioxidants are depleted in pan masala tobacco users with subsequent alteration in the biochemical parameters. Supplementation of antioxidants may prevent oxidative damage in pan masala tobacco users.

Prostate biomarkers with reference to body mass index and duration of prostate cancer.

OBJECTIVE: This study was performed to assess prostate biomarkers with reference to body mass index and duration of prostate cancer. MATERIALS AND METHODS: A hospital based retrospective study was undertaken using data retrieved from the register maintained in the Department of Biochemistry of Manipal Teaching Hospital, Pokhara, Nepal between 1st January, 2009 and 28th February, 2012. Biomarkers studied were prostate specific antigen (PSA), acid phosphatase (ACP) and prostatic acid phosphatase (PAP), alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (γGT). Demographic data including age, duration of disease, body weight, height and body mass index (BMI) were also collected. Duration of disease was categorized into three groups: <1 year, 1-2 years and >2 years. Similarly, BMI (kg/m2) was categorized into three groups: <23 kg/m2, 23-25 kg/ m2 and >25 kg/m2. Descriptive statistics and testing of hypothesis were used for the analysis using EPI INFO and SPSS 16 software. RESULTS: Out of 57 prostate cancers, serum level of PSA, ACP and PAP were increased above the cut-off point in 50 (87.5%), 30 (52.63%) and 40 (70.18%) respectively. Serum levels of PSA, ACP and PAP significantly declined with the duration of disease after diagnosis. We observed significant and inverse relation between PSA and BMI. Similar non-signficiant tendencies were apparent for ACP and PAP. CONCLUSIONS: Decreasing levels of prostate biomarkers were found with the duration of prostate cancer and with increased BMI. Out of prostate biomarkers, PSA was found to be significantly decreased with the duration of disease and BMI.

Prevalence and association of microalbuminuria in essential hypertensive patients.

BACKGROUND: Microalbuminuria in hypertension has been described as an early sign of kidney damage and a predictor for end stage renal disease and cardiovascular disease. Thus, it is of great importance to study urinary albumin creatinine ratio and progression of kidney disease in hypertensive patients. AIMS: The present study was undertaken to find out the prevalence and association of microalbuminuria in newly diagnosed essential hypertension. MATERIALS AND METHODS: Newly diagnosed essential hypertensive cases (n = 106) and normotensive controls (n = 106) were enrolled. Hypertension was defined according to Joint national committee-VII definitions. Microalbuminuria was measured using an U-Albumin (NycoCard, Norway) and adjusted for urine creatinine. Descriptive statistics and testing of hypothesis were used for the analysis using SPSS 16 software. RESULTS: 51.88% of hypertension cases and 13.2% of normotensive controls had microalbuminuria in total population (odds ratio 7.086, P-value <0.001). 46.67% of cases and 12.08% of controls had microalbuminuria in male population (odds ratio 6.375, P-value <0.001). Similarly, 58.7% of cases and 14.58% of controls had microalbuminuria in female population (odds ratio 8.32, P-value <0.001). CONCLUSIONS: By showing strong association between microalbuminuria and hypertension, our findings suggest that microalbuminuria could be a useful marker to assess risk management of cardiovascular disease and renal disease.