Citrate is an intermediate in the tricarboxylic acid cycle (TCA cycle, Krebs cycle), a central metabolic pathway for all aerobic organisms, including animals, plants, and bacteria ^[1][2][1,2]. In humans, citrate is produced in the mitochondria after the condensation of acetyl coenzyme A (acetylCoA) and oxaloacetate, which is catalysed by citrate synthase; it then enters the TCA cycle, thus becoming the primary adenosine 5′-triphosphate (ATP) provider by which living cells harvest the energy they need to accomplish essential and specific functions [1].

There is a balance between citrate availability and elimination, depending on physiological requirements. Basically, citrate homeostasis depends on four domains, i.e., nutritional intake, renal clearance, cellular metabolism, and bone remodelling (Figure 1).

Citric acid is naturally contained in fruits and vegetables, particularly in citrus fruits, with concentrations ranging from 0.005 mol/L in oranges and grapefruit to 0.30 mol/L in lemons and limes ^[6][7][7,8]. Food citrate may also be produced biotechnologically by many microorganisms through a fermentation process, and of these, Aspergillum Niger has been recognised as the most efficient producer ^[8][9]. Industrial-scale citric acid is employed instead of fresh lemon juice in a variety of pre-prepared meals, and it is also used as an additive in foods and beverages since it acts as a preservative, acidity regulator, flavouring substance and emulsifying agent. In fact, due to the variety of applications, citric acid is the most consumed organic acid in the world, leading to high commercial interest as well as motivating scientists to discover new super-producing techniques ^[9][10].

As circulating citrate is freely filtered in the glomerulus, 24-h excretion is considered to be a valid marker for highlighting alterations of citrate homeostasis ^[10][11]. The reference values for urinary citrate levels range from 320 to 1260 mg/24 h, with an average in males of 550 mg/24 h and in females of 680 mg/24 h ^[17][18][18,19]. The higher excretion of citrate in females is in relation to the estrogenic rate ^[19][20] and explains the lower incidence of nephrolithiasis in premenopausal women, considering that citrate-calcium binding is one of the main mechanisms for inhibiting stone formation ^[20][21]. Based on the reference values for lithogenic risk, the threshold for the diagnosis of hypocitraturia is usually fixed as less than 320 mg per day. However, hypocitraturia may be severe (citrate excretion of less than 100 mg per day) or mild-moderate (from 100 to 320 mg), but low excretion (less than 640 mg per day) could also be a significant sign and should be monitored ^[17][18]. In general, elevated citrate excretion may be considered a non-pathological condition which reflects the restoration of the acid-base balance and occurs, for instance, after chronic alkali intake ^[21][22]. Low citrate excretion is a relatively common finding, and even though the majority of patients have idiopathic hypocitraturia, there are several medical and physiological conditions associated with this abnormality. All the conditions listed in Table 1  may be potentially associated with bone metabolism alterations, even when there are no obvious symptoms.

The detrimental effect of acid-base imbalance on bone metabolism has been proven without a doubt ^[35][36][36,37], thus suggesting that citraturia could be a noninvasive and indirect view of bone health status. Actually, the relationship amobetweeng urinary citrate excretion, bone quality parameters, and circulating levels of bone turnover markers has been demonstrated ^[33][35][34,36], even if its clinical usefulness is still controversial ^[36][37].

In 1941 Dickens stated that approximately 90% of the total citrate found in the body of “osteovertebrates” resides in mineralised tissues, but the most valuable insight was that, due to its high binding affinity to calcium stored in the hard tissue, citrate could play a pivotal role in regulating metabolic functions and in maintaining the structural integrity of bone ^[37][6]. Moreover, Dickens postulated that the presence of citrate in bone is crucial for preventing calcium precipitation, either when bone tissue is resorbed in response to lowered serum calcium or when the biomineralisation process starts.

Over time, data in the literature regarding the relationship between citrate and bone physiology have been increasing exponentially, but the role of citrate in driving the structural and functional properties of healthy bone in humans has only been recently elucidated. Nowadays, there is adequate knowledge regarding changes in the citrate metabolism during the whole process of differentiation of the mesenchymal stem cells (MSCs) into mature osteoblasts (Figure 2) ^[5][38][39] [5,53,56], how citrate enters the crystalline structure of bone, and how it controls the size and morphology of apatite nanocrystals (Figure 3) ^{[40][41][42][43][44]}[38,40,43,44,45 ].

Taken together, studies regarding the role of citrate in the nanocrystal structure and those showing that osteoblasts were the specialised citrate-producing cells in bone have led to a new concept of bone formation related to “citration”. Briefly, Costello et al. (2012) stated that….”when considering the mineralisation role of osteoblasts in bone formation, it now becomes evident that ’citration’ must be included in the process. Mineralisation without ‘citration‘ will not result in the formation of normal bone, i.e., bone that exhibits its important properties, such as stability, strength, and resistance to fracture” ^[38][53].

Figure 2. Citrate metabolism, osteoblast differentiation, and mineralisation process. The figure combines the concept of “osteoblast citration” with the main steps of the differentiation of MSCs into bone-forming cells (osteoblasts). (A) Resting MSCs are quiescent, nonproliferating cells which exhibit the typical mitochondrial metabolism with the oxidation of citrate via the Krebs cycle. (B) In the presence of proper stimuli, the undifferentiated MSCs are committed to osteogenic differentiation and, at the early phase, high proliferation is required. To accomplish this goal, the following events are necessary: (1) the upregulation of the “zinc importer protein 1” (ZIP1), which promotes the zinc intake, (2) the accumulation of mitochondrial citrate due to the zinc-dependent inhibition of the mitochondrial aconitase, (3) the exportation of citrate into cytosol by means of the “citrate transport protein” (CTP/SLC25A1), (4) the use of cytosol citrate for the lipogenesis process which is essential for cell duplication. (C,D) The citrate exportation from cytosol to extracellular fluid starts during cell differentiation, and it is simultaneous with the synthesis and the release of amorphous CaP, collagen, and noncollagenous proteins. (E) The “osteoblast citration” is completed when the mineralised matrix is assembled. The role of citrate in growing the apatite nanocrystals and driving the mineralisation process is explained in Figure 3.

 

Figure 3. Citrate in the formation of the mineral matrix. (A) The amorphous calcium-phosphate (CaP) phase originates from an oversaturated CaP solution, and the mineralisation process starts when the organic phase (citrate, collagen fibrils, and noncollagenous proteins) is available in the bone microenvironment. (B) At the early stage, few citrate molecules bind with the amorphous CaP and the particle aggregation is slowed down. (C) In the next phase, the noncollagenous proteins released from bone cells favour CaP aggregation and apatite nucleation while the collagen promotes the self-assembly of CaP and guides the aggregate deposition on the collagen surface. (D) When the surface is fully covered by citrate, the thickness increase is inhibited (2–6 nm), while longitudinal growth continues up to 30–50 nm, thus explaining the flat morphology of bone mineral platelets. In addition, citrate forms bridges between the mineral platelets which can explain the stacked arrangement which is relevant to the mechanical properties of bone.

 

4. Citrate Pathophysiology and Bone Diseases

The role of citrate in mineralised tissues poses several questions regarding the consequences of a low bioavailability at the systemic level. For the most part, published data linking citrate alteration with bone metabolism refer to renal diseases, acid-base imbalance or also physiological conditions such as menopause, but there are also inheritable genetic defects which affect the TCA cycle in mitochondria or the citrate transport. In the following paragraphs, the medical conditions in which the association between citrate and bone health status has been implied are discussed.

4.1. Bone Health Status and Alterations of Citrate Homeostasis in Kidney Diseases

With the progressive ageing of the population, epidemiological studies have shown a higher rate of elderly-related illnesses, including the impairment of bone quality leading to osteoporosis and decreased renal function with chronic kidney disease (CKD), which in turn may influence bone health status ^[45][46][47][66,67,68]. The decrease in renal function may be mild, moderate or severe on the basis of estimated-glomerular filtration rate (GFR) equations and is associated with the simultaneous impairment of mineral homeostasis, including serum and tissue concentrations of phosphorus and calcium, circulating levels of calciotropic hormones (PTH, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D), fibroblast growth factor-23, and growth hormone. The modifications of mineral homeostasis may promote a loss of bone mass and an increase in bone fragility ^[48][69]. As observed by Malmgren et al. (2015), approximately 95% of women over 75 years of age showed a mild-moderate decrease in renal function (CKD stages 2–3) which may have had a harmful effect on bone health ^[49][70]. In a 10-year longitudinal study, the authors evaluated the long-term influence of impaired renal function on bone mineral density (BMD) ^[50][71]. They analysed 1044 Caucasian women from the “Osteoporosis Prospective Risk Assessment” (OPRA) cohort and found that renal function was positively correlated with femoral neck BMD in elderly women, although the association attenuated as ageing progressed. Women with poor renal function had a higher annual rate of bone loss over 5 years compared to those with normal function, and markers of mineral homeostasis were more frequently altered. High-throughput “omics” approaches, including metabolomics, have been proposed to identify new biomarkers which could help the management of CKD patients, and TCA cycle-metabolites are emerging as potential candidates ^[51][72]. A significantly reduced urinary excretion of citrate (–68%) has been observed in non-diabetic patients with CKD as compared to subjects with normal renal function. The renal expression of genes regulating the TCA cycle was decreased in subjects who had impaired renal function, thus suggesting that mitochondrial dysfunction could be involved in the pathogenesis of CKD ^[52][73]. Moreover, GFR positively correlated with citrate excretion, and kidney stone formers with CKD had significantly lower urinary citrate excretion than subjects with kidney stone disease and normal renal function ^[53][74]. To the researcheuthors’ knowledge, the link between CKD, urinary citrate and bone health status has still not been elucidated but, taking into account the information emerging from the previous paragraphs, it is reasonable to assume that the link exists. Some indications have derived from the data regarding kidney stone disease, which is the paradigmatic expression of a relationship between citrate alterations, BMD decrease and fracture risk that has been investigated since the 1970s ^[54][75]. In fact, several studies have shown that osteoporotic fractures occurred more frequently in patients with kidney stones than in the general population ^{[55][56][57][58]}[76,77,78,79]. The connection between kidney stones and bone metabolism is related to several factors. Briefly, kidney stones form when urine becomes supersaturated with respect to its specific components. Since 80% of kidney stones are composed of calcium-oxalate (CaOx) or CaP, the regulation of calcium excretion plays a pivotal role in the etiopathogenesis of nephrolithiasis ^[59][80]. As urinary citrate is able to bind calcium and prevent the growth and agglomeration of CaOx and CaP crystals, the close relationship between low citrate excretion and kidney stone formation has been fully established ^[10][11]. The incidence of hypocitraturia varies from 20% to 60% in people who have a propensity to form stones, either as a single abnormality or in conjunction with other metabolic disorders ^[15][16]. Hypercalciuria may occur either when filtered calcium is abnormally increased or when its reabsorption is abnormally decreased. The former may be associated with enhanced bone resorption which raises calcium bioavailability at the systemic level, while the latter may be the consequence of decreased renal function as occurs in CKD. Theoretically, reduced GFR in CKD should lead to decreased urinary calcium concentration, but the consequences of defective tubular reabsorption are more relevant and are responsible for the supersaturation of calcium salts. In addition, in the distal nephron, calcium reabsorption is a PTH-dependent process, PTH being the hormone capable of stimulating the resorption of the bone matrix in response to low, systemic calcium availability ^[60][81]. Therefore, as the decrease in the renal function progresses, PTH levels and bone loss gradually increase, thus explaining why kidney stones are a significant predictor of osteoporotic fracture in patients with CKD ^[61][82]. Moreover, when nephrolithiasis occurs, patients are frequently advised to reduce calcium intake, thus favouring a negative calcium balance which is an additional risk factor promoting a decrease in BMD ^[10][11]. Recent findings have demonstrated that lithogenic risk factors, including hypocitraturia, are also detectable in patients without kidney stones who exhibit osteoporosis or osteopenia, thus leading to the hypothesis that the evaluation of lithogenic risk could have significant implications for monitoring bone health status ^[33][62][34,83].

4.2. Postmenopausal Osteopenia and “Net Citrate Loss”

4.2. Postmenopausal Osteopenia and “Net Citrate Loss”

Estrogen deficiency and ageing are the main factors responsible for the depletion of bone mass ^[63][84], but they are also associated with changes in urine composition which are similar to those of subjects having an increased risk of kidney stones ^[10][11]. The circulating citrate levels and the citrate content in bone are markedly reduced in animals with age-related or ovariectomy-induced bone loss ^[64][85]. A low citrate excretion, less severe than true hypocitraturia fixed at less than 320 mg per day, has been described in postmenopausal women ^[10][32][11,33] and in subjects with a low bone mass ^[33][62][34,83]. Nurses’ Health Study II considered an ongoing cohort of 108,639 participants from whom information on menopause and kidney stones was obtained. In general, postmenopausal status was associated with lower BMD and a higher incidence of kidney stones in this cohort. Moreover, small but significant differences in urine composition were found in 658 participants who had pre- and postmenopausal 24-h urine analyses, including a lower citrate excretion ^[65][86]. The postmenopausal decline in estrogen concentration influences the activation rate of basic multicellular units composed of bone-resorbing osteoclasts and bone-forming osteoblasts. However, according to Drake et al., resorption increased by 90% while formation increased by only 45% ^[66][87] and the final result was a “net bone loss”. This imbalanced bone remodelling depends on the effects that the lack of estrogen has on bone cells. On the one hand, the activity of the receptor activator of the nuclear factor-κ B ligand (RANKL) is promoted, a key factor in osteoclast differentiation; on the other hand, the osteogenic precursors are destined to differentiate into adipocytes, and the survival of mature osteoblasts is suppressed ^[67][88]. The result is the reduction of mature osteoblasts, and since they are the cells capable of synthesising citrate [5], the consequence is lower citrate production which impairs the quality and the stability of the bone microarchitecture ^[40][41][38,40]. Moreover, osteoclast differentiation and bone resorption are energy-demanding processes, and the citrate which is synthesised cannot be accumulated because it is essentially utilised through the citric acid cycle ^[68][69][89,90]. Similarly, the MSC differentiation towards adipocytes requires more citrate as a source of cytosolic acetylCoA for lipid biosynthesis ^[64][85]. In conclusion, according to Granchi et al., estrogen deficiency leads to a “net citrate loss” which could explain the diminished citrate excretion observed in postmenopausal women ^[70][91].

4.3. Genetic Variations Influencing Citrate Homeostasis and Skeletal Development

4.3. Genetic Variations Influencing Citrate Homeostasis and Skeletal Development

The “Online Mendelian Inheritance in Man^®” database (OMIM^®) is a comprehensive repository of information on the relationship between genetic variation and phenotypic expression ^[71][92].  The annotations connecting citrate homeostasis with skeletal defects are listed in  Table 2, and many of these concern Slc proteins, which are a family of solute transporters through the membranes. Mutations of the Cl2/HCO3·2 exchanger AE1, encoded by SLC4A1 which is expressed in red blood cells and in type A intercalated cells of the renal collecting tubule, may be responsible for distal renal tubular acidosis (dRTA), with or without haemolytic anaemia. The corresponding phenotype displays defective urine acidification, nephrocalcinosis, nephrolithiasis, hypercalciuria, and hypocitraturia [93]. The clinical phenotype in patients with inherited dRTA is characterised by stunted growth with bone abnormalities in children, as well as nephrocalcinosis and nephrolithiasis which develop as the consequence of hypercalciuria, hypocitraturia, and relatively alkaline urine. The same cytogenetic location of SLC4A1 (17q21.31) is involved in Glycogen storage disease Ia which is caused by a deficiency in glucose-6-phosphatase activity that catalyses the synthesis of glucose from glucose-6-phosphate. This enzymopathy results in a failure to maintain normal glucose control with glycogen accumulation in the liver, kidney, and intestine. Low citrate excretion and hypercalciuria have been described by Weinstein et al. (2001), and the combination of these metabolic alterations correlated with the onset of nephrocalcinosis and nephrolithiasis ^[73][94]. Furthermore, there is increasing evidence that poor metabolic control, including chronic acidosis (lactic), low muscle mass and delayed puberty, may negatively affect BMD in half of the patients ^[74][95]. NaCT is the sodium-coupled tricarboxylate transporter predominantly expressed in the liver, at several-fold lower levels in the testis and the brain, and at weak levels in the kidney and the heart. The association between the mutations of the SLC13a5 gene on chromosome 17p13 and early infantile epileptic encephalopathy-25 with amelogenesis imperfecta has been clearly recognised ^[75][96]. More recently, Irizarry et al. have shown that SLC13a5 deficiency led to decreased BMD and impaired bone formation in homozygote (Slc13a5-/-) and heterozygote (Slc13a5+/-) mice ^[76][97]. As shown by Diaz et al. (2017), the epigenetic modulation of SLC13a5 gene may also influence skeletal development, since DNA hypermethylation and low gene expression have been found in the placenta and cord blood of infants born small-for-gestational-age and correlated with low height and weight at birth, low BMD, and low mineral content ^[77][98]. Bartter syndrome refers to a group of autosomal recessive disorders characterised by impaired salt reabsorption in the thick ascending limb of the loop of Henle with pronounced salt wasting, e.g., potassium and calcium, and hypokalemic metabolic alkalosis ^[78][99]. The antenatal variant or Bartter syndrome type I is caused by a homozygous or compound heterozygous mutation in the sodium-potassium-chloride cotransporter-2 gene ^[79][100]. The affected infants develop marked hypercalciuria and, as a secondary consequence, nephrocalcinosis and osteopenia ^[80][101]. To the best of the authors’ knowledge, low citrate excretion in patients affected by Bartter syndrome has not been described, but citrate potassium administration is able to correct biochemical alterations ^[81][82][102,103]. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal-recessive renal tubular disorder caused by mutations in claudin-16 and claudin-19, which are members of the transmembrane family proteins regulating calcium and magnesium reabsorption in the kidney ^[83][104]. Thorleifsson et al. have also identified claudin-14 as a major risk gene of hypercalciuric nephrolithiasis associated with a decrease in BMD ^[84][105]. Patients can develop hypomagnesaemia, hypercalciuria, and nephrocalcinosis, and their clinical course is often complicated by the progressive loss of kidney function. Other biochemical anomalies consist of elevated serum PTH levels before the onset of CKD, incomplete distal tubular acidosis, hyperuricemia and hypocitraturia ^[85][106]. Additional symptoms may be recurrent urinary tract infections, nephrolithiasis, polyuria, polydipsia and/or failure to thrive ^[85][106]. Amelogenesis imperfecta has also been described in some patients ^[86][107]. The human gene SLC13A2 encodes the sodium-dicarboxylate cotransporter (NaDC1) which is highly expressed in the brush-border membranes of the renal proximal tubule and intestinal cells, and reabsorbs Krebs cycle intermediates, i.e., succinate and citrate ^[87][108]. Although to date no distinctive phenotype has been linked with SLC13A2 variation in the OMIM database, Okamoto et al. (2006) have hypothesised that NaDC1 alterations could play a role in the development of kidney stones by affecting the citrate concentration in the urine ^[88][109]. The functional properties and protein expression of eight coding region variants of NaDC1 have recently been characterised; the majority of them appeared to decrease transport activity and were predicted to result in decreased citrate absorption in the intestine and kidney ^[89][110]. Even if not investigated, it is reasonable to assume that effects on bone mass may occur since these conditions influence citrate metabolism and predispose to renal stone formation as well. The mitochondrial CTP, coded by the SLC25A1 gene located on chromosome 22q11.21, is embedded in the inner membrane and determines the efflux of tricarboxylic citrate from the mitochondria to cytosol in exchange for dicarboxylic malate ^[90][111]. The high citrate concentration into cytoplasm modulates the lipid synthesis and affects glycolysis by inhibiting phosphofructokinase-1 ^[91][112]. Genetic variations of SLC25A1 mainly lead to inheritable diseases featured by alterations of the central nervous system (combined D-2- and L-2-hydroxyglutaric aciduria; OMIM ID: 615182) and skeletal muscle (congenital myasthenic syndrome-23; OMIM ID: 618197) while the presence of bone defects is less relevant. However, SLC25A1 impairment also occurs in the 22q11.2 deletion syndrome which is characterised by congenital absence of the thymus and parathyroid glands as well as cardiac, renal and eye anomalies, developmental delay, and also skeletal defects. As additional evidence of a relationship between citrate transport and bone pathophysiology, it has been shown that SLC25A1 knockout in mice causes a notable decrease in the number of osteoblasts and the amount of osteoid ^[92][113]. As mentioned above, zinc plays a crucial role in regulating the extracellular bioavailability of citrate in the formation of new mineralised matrix and, therefore, gene defects involving a zinc transporter may be involved in alterations of the citrate metabolism and bone diseases. Of these, the solute carrier 39A family (SLC39A or ZIP) controls the influx of zinc into the cytoplasm ^[39][56]. In a previous study, the authors found that SLC39A13 (ZIP13) is upregulated throughout osteogenic differentiation, and no changes were recorded during the mineralisation process ^[93][58]. SLC39A13 gene defects have been associated with low bone mass in knockout mice ^[39][56] and spondylodysplastic Ehlers-Danlos syndrome, type 3 (Phenotype MIM number 612350).