Fig 5. Extracellular citrate depends on ANK/ANKH activity.

(A) Citrate plasma concentrations in wild type (Ank^wt/wt, n = 8), heterozygous (Ank^wt/ank, n = 8) and Ank^ank/ank (n = 8) mice. Citrate concentrations in urine of wild type (Ank^wt/wt, n = 6), heterozygous (Ank^wt/ank, n = 10) and Ank^ank/ank (n = 9) mice. (C) Urine of a patient with biallelic inactivating mutations in ANKH is virtually devoid of citrate. NMR spectra of urine of a patient with biallelic pathogenic ANKH^L244S mutations (C, upper panel). A representative sex- and age-matched control urine sample contained 370 μmol/mmol creatinine (C, lower panel). Spectra are scaled on creatinine. Citrate resonates as a typical AB-system (2.98 ppm; four peaks between 2.80 and 3.05 ppm). Reference values for urinary citrate for this age group are 208–468 μmol/mmol creatinine (n = 20 healthy controls) (36). Succinate resonates as a singlet resonance at 2.66 ppm. For unknown reasons, urinary lactate was somewhat increased in urine of the patient with biallelic pathogenic ANKHL244S mutations (120 μmol/mmol creatinine; reference <75 μmol/mmol creatinine). Citrate content of tibiae (D) and femora (E) of wild type (Ank^wt/wt, n = 10), heterozygous (Ank^wt/ank, n = 10) and Ank^ank/ank (n = 10) mice. Data are expressed as mean +/- SD. Statistical significance was determined by one-way ANOVA with * p < 0.05, ** p < 0.01, *** p< 0.001, **** p < 0.0001.