Maleic acid is a biomarker for maleylacetoacetate isomerase deficiency; implications for newborn screening of tyrosinemia type 1.

Dried blood spot succinylacetone (SA) is often used as a biomarker for newborn screening (NBS) for tyrosinemia type 1 (TT1). However, false-positive SA results are often observed. Elevated SA may also be due to maleylacetoacetate isomerase deficiency (MAAI-D), which appears to be clinically insignificant. This study investigated whether urine organic acid (uOA) and quantitative urine maleic acid (Q-uMA) analyses can distinguish between TT1 and MAAI-D. We reevaluated/measured uOA (GC-MS) and/or Q-uMA (LC-MS/MS) in available urine samples of nine referred newborns (2 TT1, 7 false-positive), eight genetically confirmed MAAI-D children, and 66 controls. Maleic acid was elevated in uOA of 5/7 false-positive newborns and in the three available samples of confirmed MAAI-D children, but not in TT1 patients. Q-uMA ranged from not detectable to 1.16 mmol/mol creatinine in controls (n = 66) and from 0.95 to 192.06 mmol/mol creatinine in false-positive newborns and MAAI-D children (n = 10). MAAI-D was genetically confirmed in 4/7 false-positive newborns, all with elevated Q-uMA, and rejected in the two newborns with normal Q-uMA. No sample was available for genetic analysis of the last false-positive infant with elevated Q-uMA. Our study shows that MAAI-D is a recognizable cause of false-positive TT1 NBS results. Elevated urine maleic acid excretion seems highly effective in discriminating MAAI-D from TT1.

Fast screening of N-glycosylation disorders by sialotransferrin profiling with capillary zone electrophoresis.

Background Congenital disorders of glycosylation (CDG) are a growing group of rare genetic disorders. The most frequently used screening method is sialotransferrin profiling using isoelectric focusing (IEF). Capillary zone electrophoresis (CZE) may be a simple and fast alternative. We investigated the Capillarys™ CDT assay (Sebia, France) to screen for N-glycosylation disorders, using IEF as gold standard. Methods Intra- and inter-assay precision were established, and analyses in heparin-anticoagulated plasma and serum were compared. Accuracy was assessed by comparing IEF and CZE profiles of 153 samples, including 49 normal, 53 CDG type I, 2 CDG type II, 1 combined CDG type I and type II and 48 samples with a Tf-polymorphism. Neuraminidase-treated plasma was analysed to discriminate CDG and Tf-polymorphisms using samples of 52 subjects (25 had a confirmed Tf-polymorphism). Age-dependent reference values were established using profiles of 312 samples. Results Heparin-plasma is as suitable as serum for CDG screening with the Capillarys™ CDT assay. The precision of the method is high, with a limit of quantification (LOQ) of 0.5%. All profiles, including CDG and Tf-polymorphisms, were correctly identified with CZE. Forty-nine of 52 neuraminidase-treated samples correctly identified the presence/absence of a Tf-polymorphism. Interferences in 3/52 samples hampered interpretation. Sialo-Tf profiles were dependent of age, in particular in the first three months of age. Conclusions CZE analysis with the Capillarys™ CDT kit (Sebia) is a fast and reliable method for screening of N-glycosylation defects. Tf-polymorphisms could be excluded after overnight incubation with neuraminidase.

[Persistent, therapy-resistant conjunctivitis: consider infection with Chlamydia trachomatis]. / Persisterende therapieresistente conjunctivitis.

Conjunctivitis is a frequently diagnosed disease, usually caused by a virus. A less well-known cause is a chlamydia infection. This may result in missed diagnoses, delay of treatment and several complications. We present two cases of a persistent, therapy-resistant conjunctivitis in patients who were over 70 years of age. One patient had conjunctival follicles, characteristic of chlamydia conjunctivitis. The polymerase chain reaction tests of conjunctival samples from both patients were positive for chlamydia. Both patients and their sexual partners were treated with oral azithromycin. There was a treatment delay in both cases due to late recognition which was partially due to the older age of the patients. These cases illustrate that when a patient presents with persistent, therapy-resistant conjunctivitis, particularly if conjunctival follicles are present, chlamydial conjunctivitis should be considered and conjunctival swabs should be taken, no matter what the age of the patient.

Consideration of hypertensive retinopathy as an important end-organ damage in patients with hypertension.

Longstanding and therapy-resistant hypertension may cause cerebral, renal, cardiac and retinal end-organ damage (EOD). Retinal hypertensive abnormalities are correlated with an increased risk of cardiovascular (CV) disease in general but are not included in CV risk assessment tools. Research into prevalence and determinants of retinal organ damage, such as hypertensive retinopathy (HR), is scarce. We evaluated the prevalence of HR and the association with other signs of EOD in patients with hypertension. A retrospective observational study was performed in all hypertensive patients referred by a general practitioner to the hypertension clinic at the Diakonessenhuis, Utrecht and Zeist, the Netherlands between 2011 and 2013. A screening of risk factors, albuminuria, left-ventricular hypertrophy (LVH) and retinal fundoscopy was performed. In all, 44% (123/280) of patients referred to the clinic were diagnosed with HR, while 15 and 11% were diagnosed with LVH and microalbuminuria, respectively. Patients with isolated HR consisted of 31% of all patients. When HR was added as a form of EOD, the percentage of patients with a treatment indication increased from 3 to 14%. Patients who were already on treatment goal exhibited a high prevalence of HR (28%), warranting treatment intensification. HR is prevalent in a third of hypertensive patients referred to our clinic, and isolated HR accounts for the majority of (end-) organ damages. Fundoscopy in the evaluation of hypertension might improve the indication for therapy. Furthermore, diagnosing HR could be helpful in selecting patients with hypertension on treatment goal in need of more aggressive treatment.

Enhanced glucose cycling and suppressed de novo synthesis of glucose-6-phosphate result in a net unchanged hepatic glucose output in ob/ob mice.

AIMS/HYPOTHESIS: Leptin-deficient ob/ob mice are hyperinsulinaemic and hyperglycaemic; however, the cause of hyperglycaemia remains largely unknown. METHODS: Glucose metabolism in vivo in 9-h fasted ob/ob mice and lean littermates was studied by infusing [U-(13)C]-glucose, [2-(13)C]-glycerol, [1-(2)H]-galactose and paracetamol for 6 h, applying mass isotopomer distribution analysis on blood glucose and urinary paracetamol-glucuronide. RESULTS: When expressed on the basis of body weight, endogenous glucose production (109+/-23 vs 152+/-27 micromol.kg(-1).min(-1), obese versus lean mice, p<0.01) and de novo synthesis of glucose-6-phosphate (122+/-13 vs 160+/-6 micromol.kg(-1).min(-1), obese versus lean mice, p<0.001) were lower in ob/ob mice than in lean littermates. In contrast, glucose cycling was greatly increased in obese mice (56+/-13 vs 26+/-4 micromol.kg(-1).min(-1), obese versus lean mice, p<0.001). As a result, total hepatic glucose output remained unaffected (165+/-31 vs 178+/-28 micromol.kg(-1).min(-1), obese vs lean mice, NS). The metabolic clearance rate of glucose was significantly lower in obese mice (8+/-2 vs 18+/-2 ml.kg(-1).min(-1), obese versus lean mice, p<0.001). Hepatic mRNA levels of genes encoding for glucokinase and pyruvate kinase were markedly increased in ob/ob mice. CONCLUSIONS/INTERPRETATION: Unaffected total hepatic glucose output in the presence of hyperinsulinaemia reflects hepatic insulin resistance in ob/ob mice, which is associated with markedly increased rates of glucose cycling. Hyperglycaemia in ob/ob mice primarily results from a decreased metabolic clearance rate of glucose.

Reliability of exophthalmos measurement and the exophthalmometry value distribution in a healthy Dutch population and in Graves' patients. An exploratory study.

AIM: The purpose of this study was to test the reliability of an exophthalmometer commonly used in the Netherlands; to determine the exophthalmometry value distribution with this instrument and to assess the upper exophthalmometry limits of normal in a healthy, adult, Caucasian, Dutch population. Furthermore, to assess the effects of gender and age on exophthalmometry readings in this group and in a group of Graves' patients by comparing healthy, adult, Caucasian, Dutch individuals with adult, Caucasian, Dutch Graves' patients. METHODS: To test the reliability of our Hertel exophthalmometer, we determined the interobserver variation between two observers by measuring 160 eyes in healthy, adult, Caucasian, Dutch females and males (10 females and 10 males in each decade between 20 and 60 years of age). These data were also used for the assessment of the Hertel value distribution and for defining the upper limits of normal in these individuals by logistic regression analysis. The effects of disease, age and gender were established using these data plus data of a retrospective study of 393 adult, Caucasian, Dutch females (n=294) and males (n=99) with Graves' orbitopathy in whom Hertel values were measured with the same exophthalmometer. RESULTS: Exophthalmometry using an Hertel exophthalmometer appeared reliable (Pearson correlation coefficient for interobserver variation 0.89; 96% of the Hertel values measured by two observers were within the limits (of 2 mm) of agreement). Hertel values usually show a normal distribution in healthy individuals and in Graves' patients and are sex- and age-dependent, but there was no dependence on age in this small series in adults. Logistic regression analysis revealed an upper limit of normal of 16 mm in females and 20 mm in males in our group, using the exophthalmometer described. CONCLUSIONS: Exophthalmometry is reliable and absolute measurement of proptosis is feasible. International standardization of Hertel exophthalmometry is required in order to compare exophthalmometry data in the literature reliably.

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048.

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.

Occurrence of retained lens fragments after phacoemulsification in The Netherlands.

PURPOSE: To determine the incidence of retained lens fragments after phacoemulsification in The Netherlands and to evaluate the effect of vitrectomy on this complication. SETTING: Eleven vitreoretinal centers in The Netherlands. METHODS: We performed a retrospective analysis of all patients with retained lens fragments (N = 70) who were referred for vitreoretinal surgery to 11 specialized centers. Seven patients (10%) were treated with medication alone, and 63 (90%) had pars plana vitrectomy. Minimum follow-up after vitrectomy was 3 months. RESULTS: The incidence of retained lens fragments in The Netherlands was calculated at 0.9/1000 cataract operations. Retained lens fragments occurred during the learning curve and with experienced surgeons. After medical or surgical treatment, visual acuity was 20/40 or better in 43 of 70 patients (61%). Uveitis disappeared in all cases. Retinal detachment occurred in 10 patients (14%). Attached retinal breaks were treated in an additional 5 patients. Corneal grafting was performed in 2 patients. Patients who had immediate vitrectomy did not have better functional results than patients in whom vitrectomy was delayed. The iris-fixated claw lens was implanted successfully when capsular support was insufficient. CONCLUSIONS: The introduction of phacoemulsification in The Netherlands is associated with an increase of patients with retained lens fragments. Retained lens fragments are complicated by an increased risk for retinal detachment and corneal decompensation. Vitrectomy resulted in a marked improvement of visual acuity and clearing of uveitis.

Peribulbar corticosteroid injection: vitreal and serum concentrations after dexamethasone disodium phosphate injection.

PURPOSE: To study the dexamethasone level reached in human vitreous after a peribulbar injection of 5 mg of dexamethasone disodium phosphate and to assess its systemic uptake. METHODS: In a prospective study, 61 eyes of 61 patients scheduled for vitrectomy received a single peribulbar injection of 5 mg of dexamethasone disodium phosphate at varied intervals before surgery. At the start of vitrectomy, an undiluted vitreous sample was taken. In 22 patients, multiple serum samples were collected. Dexamethasone concentrations were measured by radioimmunoassay. The physiologic cortisol concentration was determined in the vitreous of 12 eyes of 12 patients who did not receive dexamethasone. RESULTS: An average dexamethasone peak concentration of approximately 13 ng/ml was reached in vitreous 6 to 7 hours after peribulbar injection. In serum the average peak concentration was approximately 60 ng/ml 20 to 30 minutes after peribulbar injection. The average physiologic cortisol concentration in vitreous was 5.1 ng/ml. CONCLUSIONS: After a peribulbar injection of 5 mg of dexamethasone disodium phosphate, an average intravitreal dexamethasone concentration is reached with a 75 times greater anti-inflammatory potency than physiologically present cortisol. Dexamethasone concentration in serum, however, is several times higher. Peribulbar injection is not just a local treatment but results in serum levels comparable to those achieved by a high oral dose.