The first experience of using beta-hydroxybutyrate analysis of capillary blood in the diagnosis of non-diabetic hypoglycemia in adults

Background: The diagnostic threshold of β-hydroxybutyrate (BHB) at the moment of hypoglycemia in insulinoma was developed for venous blood many years ago, when there were no alternative ways to measure ketones. Number of works, mainly on patients with diabetes mellitus, found differences in the measurement of this indicator in venous and capillary blood, but the results were contradictory. Moreover, this study was not previously used in the diagnosis of non-diabetic hypoglycemia (NDH) in adults on the territory of the Russian Federation.Aim: To estimate the effectiveness of the method for determining BHB in capillary blood and its place in the diagnosis of NDH.Materials and methods: We conducted an experimental, cross-sectional, comparative study and included patients with suspected NDH who underwent a standard fast test. The BHB level in capillary blood was determined every 6 hours during the fast test and at its completion.Results: Based on the results of the fast test, the participants (n=154) were divided into groups: with hyperinsulinemic variant of NDH and IFRoma (n=98; group 1), with hypoinsulinemic variant of NDH /absence of NDH (n=56; group 2). When comparing the level of BHB at the moment of fasting completion, significant differences were obtained between groups 1 and 2 (p<0.001). According to the ROC analysis, the determination of BHB for differentiation the hyper- and hypoinsulinemic variants of hypoglycemia is characterized by excellent quality of model (AUC=99,1% [98,0%; 100,0%]). The BHB determination in capillary blood has the maximum diagnostic accuracy at a cut-off point of ≤ 1.4 mmol/L (Se 98.0%, Sp 96.4%, PPV 98.0%, NPV 96.4%, Ac 97.4%). Exceeding the diagnostic threshold of BHB was first recorded after 24h of fasting; at the same point, a significant difference was determined when comparing BHB indicators between two consecutive measurements (between 18h and 24h).Conclusion: The BHB determination in capillary blood is a highly sensitive and highly specific additional method for the differential diagnosis of NDH variants. The diagnostic threshold for BHB of capillary blood, which allows differentiating hyper- and hypoketonemic variants of NDH, is ≤1.4 mmol / L. It is advisable to initiate control of BHB in the blood no earlier than 18 hours after the start of the fast test

Effect of glucocorticoids on bone metabolism in replacement therapy of adrenal insufficiency. Literature review

Adrenal insufficiency (AI) is a syndrome caused by disturbance in the synthesis and secretion of hormones of the adrenal cortex, which ensure the vital activity, energy and water-salt homeostasis. The widest hormonal deficiency is observed in primary hypocorticism, when the synthesis of not only glucocorticoids (GC) and adrenal androgens, but also mineralocorticoids is disrupted. Lifelong replacement therapy with GCs for this pathology may be associated with a risk of bone loss and osteoporosis. However, at present, there are no clear guidelines for diagnosis of bone condition, including and bone mineral density (BMD) monitoring during treatment with GCs in patients with AI. This review summarizes collected data on the key pathogenetic links of glucocorticoid-induced osteoporosis, incidence of decreased BMD and fractures in patients with AI. In this review factors that influence bone metabolism in this cohort of patients are considered: the type and the dose of prescribed GCs, the type (primary, secondary, HH in congenital adrenal cortex dysfunction) and the duration of AI, age, gender, and the presence of concomitant endocrine disorders (hypogonadism, growth hormone (GH) deficiency). In addition, the review presents data on the effect of adrenal androgen replacement therapy and recombinant GH therapy on bone metabolism in secondary AI

A case of 17-beta-hydroxysteroid dehydrogenase deficiency type 3 in adult endocrinologist practice

17β-Hydroxysteroid dehydrogenase 3 deficiency (17HSD3) is a rare autosomal recessive cause of 46, XY disorders of sex development resulting from HSD17B3 gene mutations, in which conversion of androstenedione to testosterone is impared. The clinical signs of 17HSD3 deficiency depend on the residual activity of the enzyme. The diagnosis of 17HSD3 deficiency is based on reduced testosterone/androstenedione ratio (T/AD < 0.8). Patients are usually assigned at birth and raise as female. If the diagnosis is made before puberty, gonadectomy is recommended, taking into account the risk of masculinization during the puberty and estrogen therapy initiation in this period. If the diagnosis of 17HSD3 deficiency is established during puberty, when virilization manifests, the therapeutic strategy is based on the results of comprehensive psychological testing and gender identity of a patient. In patients with more pronounced masculinization or diagnosis established shortly after birth, who are assigned at birth and raise as male, testosterone therapy is used to achieve a male phenotype. The 17HSD3 deficiency and virilization often result in a change of gender identity during puberty. The article presents a clinical case of 17-βhydroxysteroid dehydrogenase type 3 deficiency with late diagnosis due to parental will. The diagnostic approaches and management of the disease are also described

Comparison of Pheochromocytoma-Specific Morbidity and Mortality among Adults with Bilateral Pheochromocytomas Undergoing Total Adrenalectomy vs Cortical-Sparing Adrenalectomy

Importance: Large studies investigating long-term outcomes of patients with bilateral pheochromocytomas treated with either total or cortical-sparing adrenalectomies are needed to inform clinical management. Objective: To determine the association of total vs cortical-sparing adrenalectomy with pheochromocytoma-specific mortality, the burden of primary adrenal insufficiency after bilateral adrenalectomy, and the risk of pheochromocytoma recurrence. Design, Setting, and Participants: This cohort study used data from a multicenter consortium-based registry for 625 patients treated for bilateral pheochromocytomas between 1950 and 2018. Data were analyzed from September 1, 2018, to June 1, 2019. Exposures: Total or cortical-sparing adrenalectomy. Main Outcomes and Measures: Primary adrenal insufficiency, recurrent pheochromocytoma, and mortality. Results: Of 625 patients (300 [48%] female) with a median (interquartile range [IQR]) age of 30 (22-40) years at diagnosis, 401 (64%) were diagnosed with synchronous bilateral pheochromocytomas and 224 (36%) were diagnosed with metachronous pheochromocytomas (median [IQR] interval to second adrenalectomy, 6 [1-13] years). In 505 of 526 tested patients (96%), germline mutations were detected in the genes RET (282 patients [54%]), VHL (184 patients [35%]), and other genes (39 patients [7%]). Of 849 adrenalectomies performed in 625 patients, 324 (52%) were planned as cortical sparing and were successful in 248 of 324 patients (76.5%). Primary adrenal insufficiency occurred in all patients treated with total adrenalectomy but only in 23.5% of patients treated with attempted cortical-sparing adrenalectomy. A third of patients with adrenal insufficiency developed complications, such as adrenal crisis or iatrogenic Cushing syndrome. Of 377 patients who became steroid dependent, 67 (18%) developed at least 1 adrenal crisis and 50 (13%) developed iatrogenic Cushing syndrome during median (IQR) follow-up of 8 (3-25) years. Two patients developed recurrent pheochromocytoma in the adrenal bed despite total adrenalectomy. In contrast, 33 patients (13%) treated with successful cortical-sparing adrenalectomy developed another pheochromocytoma within the remnant adrenal after a median (IQR) of 8 (4-13) years, all of which were successfully treated with another surgery. Cortical-sparing surgery was not associated with survival. Overall survival was associated with comorbidities unrelated to pheochromocytoma: of 63 patients who died, only 3 (5%) died of metastatic pheochromocytoma. Conclusions and Relevance: Patients undergoing cortical-sparing adrenalectomy did not demonstrate decreased survival, despite development of recurrent pheochromocytoma in 13%. Cortical-sparing adrenalectomy should be considered in all patients with hereditary pheochromocytoma

СТРУКТУРНО-ФУНКЦИОНАЛЬНАЯ ПЕРЕСТРОЙКА НЕЙРОНОВ ГИППОКАМПА ПОСЛЕ ОПЕРАЦИИ ПОД АНЕСТЕЗИЕЙ СЕВОФЛУРАНОМ (экспериментальное исследование)

Hippocampus plays an important role in the cognitive mnestic functions. It coordinates emotional process, defines the intensity and specificity of behavioral, neuron and hormone reactions. Structural changes in hippocampus neurons can result in learning and memory disorders. The objective: to investigate morphometric parameters of hippocampus fields in rats after abdomen surgery with exposure to sevoflurane. Subjects and methods. The experimental research was performed using Wistar rats (n = 15). Rats from the experimental group (n = 7) had abdomen surgery with anesthesia with sevoflurane with the consequent continuous exposure to it (6 hours, 2 vol. % of sevoflurane, air flow – 1 l/min.). During 21 days rats from both groups had a number of behavioral tests. In 12 hours after the last behavioral test (on the 22-th day after the start of the experiment) the rats were decapitated with consequent brain extraction. The obtained materials were fixed in 10% neutral formalin on phosphate buffer (pH 7.4) for 24 hours minimum. Preparations of CA1, Ca2, Ca3, and Ca4 fields were studied under light microscope DM-750 (Leica, Germany) using the computer image analysis software of ImageScope M with 400-fold magnification. Results. The tests showed that cytoarchitectonics of hippocampus field was intact, pyramidal neurons had a large rounded nucleus with one, two or more distinct nucleoli. No pericellular and perivascular edema was detected. In the experimental group, all fields of the hippocampus had structural and functional changes. It manifested through both quantitative and qualitative signs of neuronal damage, especially in the Ca1 field. Segments of pyramidal neurons were disorganized, morphologically modified neurons were found: hyperchromatic shriveled neurons with no nucleus or poorly visible nucleus of the irregular shape. The appearance of morphologically changed neurons and disorganization of hippocampal layers led to changes in the width of pyramidal neurons segments. Conclusion. Morphological changes of hippocampal structures of rats after surgery and anesthesia may be the cause of cognitive functions decline during the postoperative period. Гиппокамп играет важную роль в реализации когнитивно-мнестических функций. Он координирует эмоциональные процессы, определяет величину и специфику поведенческих, нейрональных и гормональных реакций. Структурные изменения нейронов гиппокампа могут приводить к нарушениям процессов обучения и памяти. Цель: изучение морфометрических показателей полей гиппокампа крыс после операции на органах брюшной полости на фоне экспозиции севофлурана. Материал и методы. Проведено экспериментальное исследование на крысах стока Вистар (n = 15). Крысы экспериментальной группы (n = 7) подвергались операции на органах брюшной полости на фоне анестезии севофлураном с последующей его длительной экспозицией (6 ч, 2 об. % севофлурана, поток воздуха 1 л/мин). В течение 21 сут у крыс обеих групп выполняли ряд поведенческих тестов. Через 12 ч после проведения последнего поведенческого теста (22-е сут после начала эксперимента) крыс декапитировали и извлекали головной мозг. Материал фиксировали в 10%-ном нейтральном формалине на фосфатном буфере (рН 7,4) не менее 24 ч. Препараты полей СА1, СА2, СА3 и СА4 изучали под световым микроскопом ДМ-750 (Leica, Германия) с помощью компьютерной программы анализа изображений ImageScope M при увеличении в 400 раз. Результаты. В контроле цитоархитектоника полей гиппокампа не нарушена, пирамидные нейроны имели крупное округлое ядро с одним, двумя и более четкими ядрышками. Перицеллюлярного и периваскулярного отека не выявлено. В экспериментальной группе структурно-функциональная перестройка затронула все поля гиппокампа. Это проявилось появлением как количественных, так и качественных признаков повреждения нейронов, в особенности в поле СА1. Отмечена дезорганизация слоев пирамидных нейронов, определены морфологически измененные нейроны: гиперхромные сморщенные без ядер или с плохо различимым ядром неправильной формы. Появление морфологически измененных нейронов и дезорганизация слоев гиппокампа приводили к изменению ширины слоя пирамидных нейронов. Вывод. Морфологическая перестройка гиппокампальных структур крыс после операции и анестезии может лежать в основе ухудшения когнитивных функций в послеоперационном периоде.

Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activityope

ВЛИЯНИЕ ГЛОБАЛЬНОЙ ИШЕМИИ-РЕПЕРФУЗИИ ГОЛОВНОГО МОЗГА НА АКТИВНОСТЬ СУКЦИНАТДЕГИДРОГЕНАЗЫ В НЕЙРОНАХ РАЗЛИЧНЫХ СЛОЕВ НЕОКОРТЕКСА

The aim of the study was to investigate changes in activity of succinate dehydrogenase (SDH) in cytoplasm of neurons of different cortical layers in early and late reperfusion period after global cerebral ischemia in rats. Reversible global cerebral ischemia was modeled by occlusion of the brachiocephalic trunk, left subclavian artery and left common carotid artery for 10 minutes and following reperfusion during 2 or 7 days. The SDH activity in cytoplasm of neurons of II, III and V cortical layers was determined histoenzymatically. It is shown that the SDH activity in neurons of the studied cortical layers was characterized by the increased reperfusion period to the 2 days with a subsequent increased activity of the reperfusion period to the 7 days. The change in the SDH activity in cytoplasm of cortical neurons depends on the particular cerebral layer and duration of postischemic reperfusion.Целью исследования являлось изучение изменения активности сукцинатдегидрогеназы (СДГ) в цитоплазме нейронов различных слоев коры головного мозга (ГМ) в раннем и отдаленном реперфузионном периоде после глобальной ишемии ГМ у крыс. Обратимую полную глобальную ишемию ГМ моделировали окклюзией плечеголовного ствола, левой подключичной артерии и левой общей сонной артерии на 10 минут, с последующей реперфузией, длительность которой составляла 2 либо 7 суток. Гистоэнзимологически определяли активность СДГ в цитоплазме нейронов II, III и V слоев коры ГМ. Показано, что активность СДГ в нейронах изученных слоев коры ГМ характеризовалась повышением ко 2-м суткам реперфузионного периода с последующим нарастанием активности к 7-м суткам периода реперфузии. Изменение активности СДГ в цитоплазме нейронов коры ГМ зависит от принадлежности к слою коры и продолжительности постишемической реперфузии

Genetic predictors of insulin-producing pancreatic tumor

Insulinoma is the most common functioning tumor of the pancreas. Approximately 5% of its cases are associated with the multiple endocrine neoplasia syndrome type 1 (MEN1), caused by mutation in the MEN1 gene. MEN1 can be manifested by pituitary and parathyroid adenomas, pancreatic neuroendocrine tumors, tumors of the thyroid gland, adrenals, intestine, carcinoids of lungs and other organs. However, in 5–10% of the patients with clinical manifestation of this syndrome, MEN1 mutations cannot be identified. Moreover, the disease can be caused by various abnormalities (mutations, polymorphisms, etc.) in other genes. More than 30 genes, associated with insulin-producing pancreatic tumors, have been described in the literature. With a known germinal mutation, the prognosis and management of patients with insulinoma can be determined by the hereditary disease with which the tumor is associated. The article emphasizes the need to search for new genetic markers that predispose to the development of insulinoma. The necessity of extended genetic testing of patients with insulinomas is discussed, primarily of young patients with multifocal lesions, family history and associated disorders

Clinical case of factitious hypoglycemia

Hypoglycemic syndrome (HGS) is a significant decrease glucose in blood, manifested by neurological symptoms, and stopped by the introduction of glucose. Among the many causes of HGS the special place is taken by the factitious hypoglycemia, as one of the variants of Munchausen syndrome. Hypoglycemia in such cases is achieved by the intentional introduction of hypoglycemic drugs. The most commonly used medications are sulfonylurea derivatives, which are affordable, inexpensive and legal. The close collaboration of clinicians with the laboratory service plays a key role in the diagnosis of factitious hypoglycemia. Since the results of biochemical and hormonal analyzes in patients with hypoglycemia due to reception of oral hypoglycemic medications and pancreatogenous HGS are identical, the only way to differentiate these conditions is by detection of insulin secretagogue substances in the blood (or urine).The determination of oral hypoglycemic medications in cases of suspicion of artificial reception is not implemented in Russia. Factitious hypoglycemia in most cases is the diagnosis of exclusion, and its confirmation if often based on detection of medications among the personal effects of patient. This is a significant difficulty given the ethical standards. However, since 2018 we conduct in our Centre the determination of 7 oral hypoglycemic medications (glibenclamide, gliquidone, gliclazide, glimepiride, glipizide, nateglinide and repaglinide) in patient’s blood using the liquid chromatography–tandem mass spectrometry (LC-MS). This article presents a clinical case of a patient without diabetes mellitus taking glibenclamide and detection of this drug using highly selective LC-MS

Genetically determined causes of hypoglycemic syndrome in adults without diabetes

Hypoglycemic syndrome is a symptom complex that results from low blood sugar levels. In the endocrinologist practice, an insulinoma that is a small tumor in the pancreas that produces an excess amount of insulin is regarded as the main cause of hypoglycemia in patients without diabetes mellitus. Various molecular and genetic disorders develop in the insulinoma tissue, that lead to a change in the secretion of insulin and its precursors.There is often a situation when it is not possible to establish the cause of lowering blood glucose levels. In such cases, the development of hypoglycemia can result from various genetically determined enzyme, autoimmune and receptor disorders that cause a change in glucose metabolism or the synthesis/bioactivity of insulin. In the mild course of such congenital diseases, hypoglycemic conditions may first manifest in adulthood.The review describes various genetic predictors (mutations) that play a decisive role in the development of enzyme, autoimmune, receptor and proliferative disorders and, as a consequence, hypoglycemia