Erythropoietin is a signaling glycoprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domains. Here we describe the total synthesis of homogeneous erythropoietin with consensus carbohydrate domains incorporated at all of the native glycosylation sites. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild-type primary sequence, themselves obtained by solid-phase peptide synthesis. The glycopeptidyl constructs were joined by chemical ligation, followed by metal-free dethiylation, and subsequently folded. This homogeneous erythropoietin glycosylated at the three wild-type aspartates with N-linked high-mannose sialic acid–containing oligosaccharides and O-linked glycophorin exhibits Procrit-level in vivo activity in mice.

http://science.sciencemag.org/content/sci/342/6164/1357.full.pdf

Erythropoietin derived by chemical synthesis.

http://www.jbc.org/content/267/11/7703.full.pdf

Role of sugar chains in the expression of the biological activity of human erythropoietin.

Objective To determine the activity of recombinant feline erythropoietin (rfEPO) in murine bioassays and evaluate its efficacy and safety in cats with erythropoietin-dependent nonregenerative anemia. Animals 26 cats (group 1, 19 cats with anemia attributed to chronic kidney disease [CKD]; group 2, 7 cats with CKD and recombinant human erythropoietin [rhEPO]-induced red cell aplasia [RCA]). Procedure The rfEPO was synthesized by use of Chinese hamster ovary (CHO) cells transfected with feline erythropoietin complementary DNA. Preclinical assessments of rfEPO included an erythroid cell proliferation assay and measurements of reticulocytosis in Balb/C mice. Clinical assessments of cats included hematologic, biochemical, and clinical examinations during 12 (group 1) or 6 (group 2) months of rfEPO treatment. Results Biological activity of rfEPO was broadly equivalent to rhEPO in preclinical murine bioassays. Median Hct and absolute reticulocyte count in cats increased significantly during the first 3 weeks of rfEPO treatment, and median Hct generally could be maintained within a target range of 30% to 40% with periodic adjustments of rfEPO doses. Unexpectedly, 5 cats in group 1 and 3 cats in group 2 that initially responded to rfEPO treatment again developed anemia that was refractory to additional rfEPO treatments, even at higher doses. Conclusions and clinical relevance Treatment with rfEPO can reestablish active erythropoiesis in most cats with CKD, even those with anemia attributable to rhEPO-induced RCA. Unfortunately, development of RCA during treatment with CHO cell-derived recombinant erythropoietin proteins was not eliminated as a serious safety concern, even for this feline-specific preparation.

Expression, bioactivity, and clinical assessment of recombinant feline erythropoietin

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 121-284% for the brilliant cresyl blue method, 141-308% for the selective hemolysis method and 85-197% for the flow cytometry method Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 59-142%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products

http://www.scielo.br/pdf/bjmbr/v36n11/5002.pdf

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Oral Administration of Recombinant Human Erythropoietin in Liposomes in Rats: Influence of Lipid Composition and Size of Liposomes on Bioavailability

A new method of in vivo bioassay for erythropoietin (EPO) is described. This method is based on the measurement of immature reticulocytes in EPO-treated mice using an automatic microcell counter, and is simpler and more precise than the existing methods of polycythaemic mouse assay and starved rat assay. Normal mice were injected subcutaneously for 3 successive days with EPO at the doses between 0 and 9.6 IU/mouse. On the following day, 20 microliters of peripheral blood from each EPO-treated mouse was collected and haemolysed with a stromatolysing agent. Quicklyser. Residual particles derived from immature reticulocytes in the stromatolysed blood cells were counted using a microcell counter (Sysmex CC-180A) and a cell monitor (Sysmex CM-5). The number of the residual particles increased in a dose-dependent manner in EPO-treated mice. The mean of correlation coefficients of five log dose-response lines was 0.924, and the mean of precision indices was 0.138. A good correlation was also observed between the residual particle counts and reticulocyte counts obtained from smears.

Simple in vivo bioassay for erythropoietin

Iron is specifically important to athletes, and attention has grown to the association between sports performance and iron regulation in the daily diets of athletes. The study presents new insights into stress, mood states, fatigue, and sweating behavior among the non-anemic athletes with sweating exercise habits who consumed a routine low dose (3.6 mg/day) of iron supplementation. In this double-blind, randomized, placebo-controlled, parallel-group study, both non-anemic male (N = 51) and female (N = 42) athletes were supplemented either with a known highly bioavailable iron formulation (SunActive® Fe) or placebo during the follow-up training exercise period over four weeks at their respective designated clinical sites. The effect of oral iron consumption was examined on fatigue, stress profiles, as well as the quality of life using the profile of mood state (POMS) test or a visual analog scale (VAS) questionnaire, followed by an exercise and well-being related fatigue-sweat. Also, their monotonic association with stress biomarkers (salivary α-amylase, salivary cortisol, and salivary immunoglobulin A) were determined using spearman's rank correlation coefficient test. Repeated measure multivariate analysis of variance (group by time) revealed that the total mood disturbance (TMD) score was significantly lower (P = 0.016; F = 6.26) between placebo and iron supplementation groups over the four weeks study period among female athletes. Also, a significant reduction in tired feeling/exhaustion after the exercise (P = 0.05; F = 4.07) between the placebo and iron intake groups was noticed. A significant within-group reduction (P ≤ 0.05) was noticed in the degree of sweat among both male and female athletes after 2 and 4 weeks of iron supplementation, while athletes of the placebo intake group experienced a non-significant within-group reduction in the degree of sweat. Overall, the result indicates routine use of low dose (3.6 mg/day) iron supplementation is beneficial for non-anemic endurance athletes to improve stress, mood states, subjective fatigue, and sweating conditions. 

https://doi.org/10.1016/j.conctc.2023.101084

Influence of iron supplementation on fatigue, mood states and sweating profiles of healthy non-anemic athletes during a training exercise: A double-blind, randomized, placebo-controlled, parallel-group study

Abstract Previous studies have examined the effects of n-3 fatty acid intake in supplement form or fish oil capsules, but there are few studies based on other foods. Perilla oil is a traditional Japanese seed oil rich in n-3 fatty acids. This randomised trial aimed to determine the appropriate n-3 fatty acid dose through consumption of perilla oil, which improves gut function and microbiota in trained athletes, and the amount of fat fuel required to provide energy to athletes involved in high-intensity training to improve athletic performance. Thirty-six female athletes training six times per week were randomly assigned to three groups according to perilla oil intake: 9 g/d (high oil intake (HOI)), 3 g/d (low oil intake (LOI)) and placebo-supplementation (PLA) groups. The HOI and LOI groups had perilla oil-containing jelly and the PLA group had placebo jelly for 8 weeks. Gut microbiota, constipation score and urinary biochemical index were measured pre- and post-intervention. The spoilage bacteria, Proteobacteria, significantly decreased (P = 0·036, d = 0·53), whereas Butyrate-producing bacteria, Lachnospiraceae, significantly increased (P = 0·007, d = 1·2) in the HOI group. Urinary indoxyl sulphate significantly decreased in the HOI group only (P = 0·010, d = 0·82). Changes in the constipation score were significantly lower in the HOI group (P = 0·020) and even lower in the LOI group (P = 0·073) than in the PLA group; there were significant differences between groups (P = 0·035). Therefore, perilla oil intake may improve gut function and microbiota in athletes, with higher doses resulting in further improvement.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/4454BF1820E2817964F615C79AB9D89C/S0007114522001805a.pdf/div-class-title-effect-of-8-week-intake-of-the-span-class-italic-n-span-3-fatty-acid-rich-perilla-oil-on-the-gut-function-and-as-a-fuel-source-for-female-athletes-a-randomised-trial-div.pdf

Effect of 8-week intake of the n-3 fatty acid-rich perilla oil on the gut function and as a fuel source for female athletes: a randomised trial

This study shows the effect of omega-3 unsaturated fatty acids via perilla oil intake on gut microbiota and constipation. Eight sedentary females participated in a nonrandomized placebo-controlled pilot study consisting of eight-week perilla oil (OIL) and placebo (PLA) intervention phases. There was a 10-month washout period between phases. All participants received 9 g of perilla oil-containing jelly in the OIL phase, and a placebo jelly in the PLA phase. Gut microbiota, α-diversity, and constipation scores were measured pre- and post-intervention in both phases. The α-diversity, an important indicator of gut microbiota diversity, was significantly increased post-intervention (4.5 ± 0.1) compared to pre-intervention (3.8 ± 0.3) in the OIL only (p = 0.021). Notably, the level of α-diversity was maintained even after the washout period of 10 months. Butyrate-producing bacteria, Lachnospiraceae (%), did not change in the OIL but were significantly reduced post-intervention (15.1 ± 4.8) compared to pre-intervention (20.1 ± 7.0) in the PLA (p = 0.040). In addition, the constipation scores were significantly or tended to be reduced during the OIL phase only (p < 0.05, p < 0.1). In conclusion, an eight-week perilla oil supplementation may enhance and establish the diversity of gut microbiota, which may relieve constipation.

/pdf/perilla-oil-an-omega-3-unsaturated-fatty-acid-rich-oil-34spmbve.pdf

Perilla Oil, An Omega-3 Unsaturated Fatty Acid-Rich Oil, Enhances Diversity of Gut Microbiota and May Relieve Constipation in Sedentary Healthy Female: A Nonrandomized Placebo-Controlled Pilot Study

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Simple in vivo bioassay for erythropoietin

Influence of iron supplementation on fatigue, mood states and sweating profiles of healthy non-anemic athletes during a training exercise: A double-blind, randomized, placebo-controlled, parallel-group study

Effect of 8-week intake of the n-3 fatty acid-rich perilla oil on the gut function and as a fuel source for female athletes: a randomised trial

Perilla Oil, An Omega-3 Unsaturated Fatty Acid-Rich Oil, Enhances Diversity of Gut Microbiota and May Relieve Constipation in Sedentary Healthy Female: A Nonrandomized Placebo-Controlled Pilot Study