Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

STUDY QUESTION: Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? SUMMARY ANSWER: VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. WHAT IS KNOWN ALREADY: Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. STUDY DESIGN, SIZE, DURATION: Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). PARTICIPANTS/MATERIALS, SETTING, METHODS: Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development. MAIN RESULTS AND THE ROLE OF CHANCE: Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Due to the nature of this work, embryo development and implantation was only examined in the mouse. WIDER IMPLICATIONS OF THE FINDINGS: The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare.

Nosocomial Infection Among Burn Patients Admitted to a Tertiary Care Hospital of Bangladesh: A Cross-Sectional Study.

Nosocomial infection is a major challenge for the appropriate management of burns. The present study aimed to investigate incidence, risk factors, and causative organisms of nosocomial infection in burn patients of Khulna, Bangladesh. This cross-sectional study was conducted among patients admitted to the Burn and Plastic Surgery Department of Khulna Medical College Hospital (KMCH) from January to December 2020. Relevant data were collected from the patients' hospital records. Samples of wound swabs and blood were collected and cultured in the microbiology laboratory of KMCH. Logistic regression models were used to determine risk factors for infective complications in burn patients. All statistical analyses were carried out using SPSS version 26.0. A total of 100 burn patients were included. Mean age was 29.2 years with a male-female ratio of 1.3:1. Flame burns were most prevalent among the patients (41%), followed by scald (23%) and electric burns (15%). Almost 40% patients had full thickness burn. The incidence of nosocomial infection was 42% (wound infection 33% and septicemia 9%). Total body surface area of burn >40% (OR 7.56, 95% CI 2.89-19.81), full thickness burn (OR 34.40, 95% CI 3.25-97.14) and prolonged hospital stay (aOR 1.31, 95% CI 1.15-1.51) were significant risk factors for nosocomial infection. Staphylococcus aureus was the most commonly isolated organism (45%), followed by Streptococcus (24%), Pseudomonas aeruginosa (19%) and Escherichia coli (12%). As the epidemiology of nosocomial infection is not the same in different health facilities, a facility-based comprehensive burn management protocol considering the local epidemiology and causative organisms of burn wound infection is crucial for the prevention and management of nosocomial infections in burn patients.

Les infections nosocomiales sont une préoccupation majeure du traitement bien conduit des brûlés. Cette étude a eu pour but d'évaluer l'incidence, les facteurs de risque de survenue et les bactéries isolées d'infections nosocomiales survenues dans le CTB de Kulna (Bangladesh). Elle a étudié les dossiers l'ensemble des 100 patients admis dans le CTB du CHU de Kulna en 2020. Les analyses bactériologiques ont été réalisées dans le laboratoire du CHU. Une régression logistique a été utilisée pour déterminer les facteurs de risque d'infection. Toutes les analyses statistiques ont été réalisées avec SSPS 26.0. L'âge moyen était de 29,2 ans, le sex-ratio de 1,3H/1F. Les flammes représentaient 41% des causes, les liquides 23% et l'électricité 15%. Quasiment 40% des patients avaient des brûlures profondes. L'incidence des accidents infectieux était de 42% (cutanée 33%, bactériémies 9%). Les facteurs de risque indépendants de survenue d'une infection étaient une atteinte sur >40 % SCT (OR 7,56; IC95 2,89-19,81), une brûlure profonde (OR 34,40 ; IC95 3,25-97,14) et un séjour prolongé (OR 1,31; IC95 1,15-1,51). Les quatre bactéries les plus fréquentes étaient S. aureus (45%), Streptococcus spp (24%), P. æruginosa (19%), et E. coli (12%). Les épidémiologies bactériennes variant selon les services d'où elles sont issues, c'est sur l'épidémiologie locale que doivent se centre les mesures de contrôle des infections nosocomiales.

Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells.

AIMS/HYPOTHESIS: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge. METHODS: We used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors. This approach avoids the use of feeders, stroma cells or serum, all of which can interfere with experimental outcomes and could preclude future clinical applications. RESULTS: This study reveals, for the first time, that activin/TGF-ß signalling blocks pancreatic specification induced by retinoic acid while promoting hepatic specification in combination with bone morphogenetic protein and fibroblast growth factor. Using this knowledge, we developed culture systems to differentiate human pluripotent stem cells into near homogenous population of pancreatic and hepatic progenitors displaying functional characteristics specific to their natural counterparts. Finally, functional experiments showed that activin/TGF-ß signalling achieves this essential function by controlling the levels of transcription factors necessary for liver and pancreatic development, such as HEX and HLXB9. CONCLUSION/INTERPRETATION: Our methods of differentiation provide an advantageous system to model early human endoderm development in vitro, and also represent an important step towards the generation of pancreatic and hepatic cells for clinical applications.

Human chorionic gonadotrophin regulates FGF2 and other cytokines produced by human endometrial epithelial cells, providing a mechanism for enhancing endometrial receptivity.

BACKGROUND: Preimplantation cross-talk between a functional blastocyst and the endometrium is critical for successful blastocyst implantation. This interaction is mediated in part by endometrial cytokines/growth factors secreted by glandular epithelium into the uterine cavity. Recent evidence suggests that blastocyst-derived hCG may influence the endometrial milieu in conception cycles thereby enhancing receptivity and implantation success. This study investigated the effect of hCG on the secretory profile of a select cohort of 44 cytokines/growth factors from primary human endometrial epithelial cells (hEECs). These factors included those with both known and unknown roles during receptivity and implantation. The expression of one previously unknown hCG-regulated factor, fibroblast growth factor 2 (FGF2), in human endometrium and its effects on hEEC function were further examined. METHODS: hEECs isolated from endometrial biopsies collected from fertile cycling women (n = 15) were treated ± recombinant hCG (0.2-20 IU/ml) for 48 h and conditioned media was quantitatively analysed using Luminex™ multiplex technology. FGF2 was further investigated by immunohistochemistry, western blot and cell-adhesion assays. RESULTS: Of 44 cytokines/growth factors examined, 39 were produced by hEECs with a distinct profile. hCG (2 IU/ml) significantly increased the production of six factors, including those with known roles in receptivity and trophoblast function (interleukin-11), blastocyst migration and adhesion (CXCL10), blastocyst development (granulocyte macrophage colony-stimulating factor) and one unknown with respect to receptivity and implantation (FGF2). Up-regulation of known hCG-regulated proteins, vascular endothelial growth factor and leukaemia inhibitory factor, validated this study. Immunoreactive epithelial FGF2 increased across the menstrual cycle, being highest in secretory and first trimester pregnancy endometrium in vivo. FGF2 (100 ng/ml) stimulated phosphorylation of ERK1/2 in hEEC with no effect on ERK1/2 abundance and stimulated hEEC adhesion to fibronectin and collagen IV (components of blastocyst/trophectoderm extracellular matrix). CONCLUSIONS: These findings clearly support roles for hCG and FGF2 in the blastocyst-endometrial cross-talk important for endometrial receptivity and blastocyst implantation.

Double blind, randomised, placebo-controlled trial to evaluate the efficacy of esomeprazole to treat early onset pre-eclampsia (PIE Trial): a study protocol.

INTRODUCTION: Pre-eclampsia is a major complication of pregnancy, globally responsible for 60 000 maternal deaths per year, and far greater numbers of fetal losses. There is no definitive treatment other than delivery. A drug that can quench the disease process could be useful to treat early onset pre-eclampsia, as it could allow pregnancies to safely continue to a gestation where fetal outcomes are significantly improved. We have generated preclinical data to show esomeprazole, a proton pump inhibitor used for gastric reflux, has potent biological effects that makes it a worthwhile therapeutic candidate. Esomeprazole potently decreases soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin secretion from placenta and endothelial cells, and has biological actions to mitigate endothelial dysfunction and oxidative stress. METHODS AND ANALYSIS: We propose undertaking a phase II, double blind, randomised controlled clinical trial to examine whether administering 40 mg esomeprazole daily may prolong gestation in women with early onset pre-eclampsia. We will recruit 120 women (gestational age of 26+0 to 31+6 weeks) who will be randomised to receive either esomeprazole or an identical placebo. The primary outcome will be the number of days from randomisation to delivery. Secondary outcomes include maternal, fetal and neonatal composite and individual outcomes. Maternal outcomes include maternal death, eclampsia, pulmonary oedema, severe renal impairment, cerebral vascular events and liver haematoma or rupture. Neonatal outcomes include neonatal death within 6 weeks after the due date, intraventricular haemorrhage, necrotising enterocolitis and bronchopulmonary dysplasia. We will examine whether esomeprazole can decrease serum sFlt-1 and soluble endoglin levels and we will record the safety of esomeprazole in these pregnancies. ETHICS AND DISSEMINATION: This study has ethical approval (Protocol V.2.4, M14/09/038, Federal Wide assurance Number 00001372, IRB0005239), and is registered with NHREC (ID 3649) and the Pan African Clinical Trial Registry (PACTR201504000771349). Data will be presented at international conferences and published in peer-reviewed journals.

Transcription factors E2F1 and E2F3 are expressed in placenta but do not regulate MMP14.

INTRODUCTION: Preeclampsia is a serious complication of pregnancy for which there are no efficacious medical treatments. Soluble endoglin is as an anti-angiogenic factor that contributes to the pathogenesis of the disease, however little is known about its molecular regulation in placenta. Recent data has demonstrated E2F transcription factors directly regulate MMPs in metastatic disease. Of particular interest was the capacity of E2F1 and E2F3 to up-regulate MMP14, a protease that cleaves and releases soluble endoglin from placenta. The aim of this study was to characterize E2F1 and E2F3 in preeclamptic placenta and assess whether silencing affects soluble endoglin release. METHODS: E2F1 and E2F3 mRNA, protein expression and localization were assessed in severe early onset preeclamptic and preterm control placentas (delivered <34 weeks gestation). E2F siRNA was administered to primary trophoblast and primary endothelial cells and effect on MMP14 mRNA expression and soluble endoglin secretion assessed. RESULTS: E2F1 and E2F3 were localized to the syncytiotrophoblast. E2F1 was significantly down regulated in severe preeclamptic placentas, while E2F3 was unchanged. Silencing E2F1 did not decrease MMP14 expression in primary trophoblast or endothelial cells. However, E2F1 silencing resulted in a significant increase in soluble endoglin secretion from both cell types, and silencing of E2F3 also significantly increased soluble endoglin release from primary trophoblast. DISCUSSION: This study demonstrates that E2F1 and E2F3 are present within the syncytiotrophoblast of placenta and that E2F1 is reduced in preeclampsia. Although silencing of either E2F1 or E2F3 does not alter MMP14 expression, both appear to regulate soluble endoglin release.

A molecular study of EBV DNA and p53 mutations in nasopharyngeal carcinoma of Saudi Arab patients.

Tumor biopsies obtained from 25 Saudi Arab patients with nasopharyngeal carcinoma (NPC) were examined for the presence of Epstein-Barr virus (EBV) DNA detected by the polymerase chain reaction (PCR) and for the incidence of p53 mutations screened by a combination of PCR, single strand conformation polymorphism (SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP). DNA sequencing was carried out to confirm the occurrence of p53 mutation. While 92% of the tumor specimens were found to carry EBV DNA, only 1/25 showed the incidence of a homozygous mutation at codon 248 of the p53 gene. The data showed that despite a high association of EBV infection with Saudi NPC, the frequency of p53 mutations was very low. Our results are consistent with the worldwide observation of infrequent p53 mutations in NPC.

Systemic administration of glycosaminoglycan polysulphate (arteparon) provides partial protection of articular cartilage from damage produced by meniscectomy in the canine.

Bilateral medial meniscectomy was undertaken in 14 mature beagles. Another two underwent arthrotomy (sham-operated controls). One week after surgery, six of the meniscectomised animals were administered glycosaminoglycan polysulphate (Arteparon) (2 mg/kg) subcutaneously three times a week for 3 weeks, then twice weekly until killed 23 weeks later. Two months before death all animals were given Na2(35)SO4 (1.0 mCi/kg) intravenously. At autopsy, articular cartilage (AC) from the medial and lateral compartments, as well as from the femoral trochlear groove and femoral head, was sampled. Proteoglycans (PGs) were isolated by 4.0 M Guanidine hydrochloride (GuHCl) extraction of AC and purified by ultracentrifugation. The PG monomers were assayed for hexuronic acid, protein, and hexosamines (galactosamine/glucosamine), and their ability to aggregate. The results indicated that Arteparon provided some protective effect to AC in the meniscectomised compartment as demonstrated histologically by reduced surface fibrillation, diminished chondrocyte cloning, and maintenance of alcianophilia. The levels of PGs and hexuronate-protein ratios in medial AC of drug-treated meniscectomised animals were found to be comparable to sham controls, whereas these parameters in the nondrug-treated meniscectomized group were depressed.

The response of the canine intervertebral disc to immobilization produced by spinal arthrodesis is dependent on constitutional factors.

Posterior lumbar spinal fusion was performed on five mature greyhounds. Two months prior to death, all of the surgical and five age-matched control greyhounds were given Na2(35)SO4 (1.0 mCi/kg) intravenously. All fusion animals were killed 6 months postoperation, and discs beneath the fusion mass as well as those adjacent to it (parafusion discs) were sampled separately and dissected into the nuclei pulposi and annuli fibrosi (AF). Proteoglycans (PGs) were extracted with 4.0 M GuHCl and then purified by CsCl density gradient ultracentrifugation. These PG monomers were subjected to Sepharose CL-2B chromatography, and their hydrodynamic size and ability to aggregate were determined. The level, extractability, and hydrodynamic size of PGs in the AF of fusion discs were found to be greater than those in control discs, as were the keratan sulfate core protein complexes prepared by chondroitin ABC lyase digestion. The ability of the 60-day-old PG subunit populations, isolated from fusion discs, to aggregate was also higher than controls. There was, however, no difference between the galactosamine/glucosamine, galactosamine/protein, glucosamine/protein, or hexuronate/protein ratios of PGs in fusion and control discs.

Soluble endoglin production is upregulated by oxysterols but not quenched by pravastatin in primary placental and endothelial cells.

INTRODUCTION: Preeclampsia is a serious pregnancy complication. Soluble endoglin (sEng) is released from the placenta and contributes to the maternal endothelial dysfunction seen in preeclampsia. Recently oxysterols, which activate the Liver X Receptor (LXR), have been implicated in producing sEng, by upregulating matrix metalloproteinase-14 (MMP14; cleaves endoglin to produce sEng) and down-regulating tissue inhibitor of metalloproteinase-3 (TIMP-3; inhibitor of MMP14). The functional experiments in that study were performed on JAR cells (human choriocarcinoma cell line) and placental explants. METHODS: We characterized LXR in severe preeclamptic placentas, and assessed whether oxysterols increase release of sEng from primary human umbilical vein endothelial cells (HUVECs), primary trophoblasts and placental explants. Given pravastatin is thought to block oxysterol production and inhibit the LXR, we examined whether pravastatin reduces sEng release. RESULTS: LXRα and ß were localized to the syncytiotrophoblast and villous tips and were significantly up-regulated in preeclamptic placenta. Oxysterols upregulated sEng production in HUVECs and placental explants although the increases were far more modest than that recently reported. Oxysterols did not upregulate sEng in primary trophoblasts. Furthermore, mRNA expression of MMP14 and TIMP-3 were not altered by oxysterols in any tissue. Surprisingly, pravastatin did not decrease oxysterol-induced upregulation of sEng. DISCUSSION: LXR is up-regulated in preeclamptic placenta. Oxysterols upregulate sEng production from human tissues, but the increase is modest, suggesting this may not be the main mechanism for the very significant elevations in sEng seen in preeclampsia. Pravastatin does not decrease sEng production. CONCLUSION: Oxysterols modestly up-regulate sEng production which is not quenched by pravastatin.

Placental SEMA3B expression is not altered in severe early onset preeclampsia.

Recent evidence suggests that Semaphorin 3B (SEMA3B) is upregulated in severe preeclampsia, and a major driver of cytotrophoblast aberrations in this disease. Here we independently assess whether SEMA3B expression is altered in a large cohort of severe early onset preeclamptic placentas. We demonstrate that SEMA3B relative mRNA expression and copy number are not changed in PE placentas. We confirm this at the protein level by western blot. Interestingly, exposure of term trophoblasts or explants to hypoxia induced a significant down regulation of SEMA3B mRNA, but a trend towards increased SEMA3B protein expression. We conclude that SEMA3B mRNA and protein is not altered in severe early onset preeclamptic placentas.

Stability of absolute copy number of housekeeping genes in preeclamptic and normal placentas, as measured by digital PCR.

Measuring mRNA expression is fundamental to placental research. Ideally, mRNA transcript numbers are directly quantified. However, PCR analysis using the ΔΔCT method relies on the stability of housekeeping genes and only reports relative expression. Digital PCR (dPCR) directly quantifies mRNA copy number and is more accurate than quantitative PCR. We quantified absolute mRNA copy number of housekeeping genes in normotensive pre-term (n = 20), severe preeclamptic (n = 11) and term (n = 12) placenta using dPCR. Whilst there was some variation, we confirm absolute mRNA copy number of GAPDH, TOP1, CYC1 and YWHAZ in placenta does not significantly alter between these cohorts, or across gestation.

Uterine lavage or aspirate: which view of the intrauterine environment?

Fluid within the uterine cavity provides the microenvironment for preimplantation blastocyst development and early implantation. Analysis of uterine fluid sampled by aspiration or lavage provides a view of this microenvironment but the similarity or otherwise of the sample components is not known. This study compared proteins in aspirates versus lavage samples taken sequentially from the same women, using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), multiplex cytokine assays, and an activity assay for proprotein convertase 6. Both lavage and aspiration enabled analysis of uterine fluid components, but they provided substantially different protein profiles. Although there were many similarities in overall protein profiles and most specific proteins examined were detected in both fluids, these were neither qualitatively nor quantitatively comparable within each participant. A likely explanation is that lavage samples the entire uterine cavity including washing the endometrial surface (glycocalyx), whereas aspiration sampling is very local.

Analysis of fertility-related soluble mediators in human uterine fluid identifies VEGF as a key regulator of embryo implantation.

Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the midsecretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen contains important mediators that modulate the endometrium and support the conceptus during implantation. This study aimed first to identify the growth factor and cytokine profile of human uterine fluid from fertile women during the midproliferative (MP; nonreceptive) and MS (receptive) phases of the cycle, and from women with unexplained infertility during the MS phase. The second aim was to determine important functions of endometrial secretions for embryo implantation. Analysis of uterine fluid using quantitative Luminex assays revealed the presence of over 30 cytokines and growth factors, of which eight [platelet-derived growth factor-AA, TNF-B, soluble IL-2 receptor-A, Fms-like tyrosine kinase 3 ligand, soluble CD40 ligand, IL-7, interferon-A2, and chemokine (C-X-C motif) ligand 1-3] were previously unknown in human uterine fluid. Comparison of the fertile MP, MS, and infertile MS cohorts revealed vascular endothelial growth factor (VEGF) levels are significantly reduced in uterine fluid during the MS phase in women with unexplained infertility compared with fertile women. Functional studies demonstrated that culturing mouse embryos with either MS-phase uterine fluid from fertile women or recombinant human VEGF significantly enhanced blastocyst outgrowth. Furthermore, treatment of human endometrial epithelial cells with uterine fluid or recombinant human VEGF-A significantly increased endometrial epithelial cell adhesion. Taken together, our data support the concept that endometrial secretions, including VEGF, play important roles during implantation. Identifying the soluble mediators in human uterine fluid and their actions during implantation provides insight into interactions essential for establishing pregnancy, fertility markers, and infertility treatment options.

Effect of diabetic state and related disorders on the urinary excretion of magnesium and zinc in patients.

We examined the urinary excretion of magnesium and zinc in 175 diabetics [19 insulin-dependent (type I) and 156 insulin-independent (type II)] and in 160 control subjects of the same origin by determining the ratio of the concentration of each of these metals to that of creatinine (creat). Electrothermal atomic absorption spectrophotometry was used for analyzing Mg and Zn in urine. There was no significant difference in the urinary excretion of Mg between the control group [mean (SEM) = 362.6 (15.1) mmole of Mg/mole of creat] and the overall [350.2 (15.7) mmole Mg/mole creat], type I [368.4 (45.0) mmole Mg/mole creat], or type II [347.9 (16.7) mmole Mg/mole creat] diabetics regardless of the disorders associated with diabetes (cardiovascular diseases, neuropathy, retinopathy, infections, and hepatic disease). In contrast, diabetics of both types [2.67 (0.14) mmole Zn/mole creat] with or without a diabetes associated disorder excreted significantly (p = 0.031 to 0.0000) more Zn than did the control subjects [1.76 (0.09) mmole Zn/mole creat]. There was positive correlation between hemoglobin A1c and urinary loss of Mg (p = 0.013) or Zn (p = 0.0241) in patients with type II diabetes. From these data, it appears that of the two elements examined only Zn is associated with higher urinary loss in diabetic state. The discrepancy between our results and those of previous studies for Mg may be ascribed to dissimilarities in the diet habits and metabolism of Mg among diabetics of different geographical origins.

Urinary excretion of chromium, copper, and manganese in diabetes mellitus and associated disorders.

The urinary excretion of chromium, copper and manganese was determined in 185 diabetics and in an equal number of control subjects by measuring the concentration of each of these metals using electrothermal atomic spectrophotometry and dividing the values by the urinary concentration of creatinine (creat) in each subject. The mean (SEM) values for the overall diabetics and the control group were 2.32 (0.17) and 2.62 (0.22) mumol Cr/mole of creat, 76.5 (5.5) and 73.9 (6.1) mumol Cu/mole of creat, and 3.56 (0.44) and 2.66 (0.3) mumol Mn/mole of creat, respectively. There was no correlation between the urinary excretion of any of the metals examined and age or sex of either group. While the cardiovascular or ophthalmologic diseases associated with diabetes did not influence the excretion of any of these metals, significantly higher urinary excretion of Cu was exhibited by diabetics with neuropathy (p < 0.0027) or infections (p < 0.014) than by those without. Also, diabetics with liver disorders or those who were not treated with insulin excreted significantly more Mn than did their diabetic counterparts.