Cobalamin is a water-soluble, cobalt-containing B vitamin [1] resulting from microbial synthesis, mainly bacteria, present in the rumen and intestine, as well as soil [2]. This vitamin is an essential catalyzer for nucleic acid synthesis and hematopoiesis [3]. In mammalian species, only two enzymes are known to be cobalamin-dependent: (i) methionine synthase, a methyl transferase, and (ii) methylmalonyl-CoA mutase, an isomerase. Methionine synthase catalyzes the transfer of a methyl group to S-adenosyl-homocysteine, generating S-adenosyl-methionine and hence methionine, while methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA, which then enters the citric acid cycle ^[4][5][6] (Figure 1) [4,5,6]. The major food sources of cobalamin are animal products; plant products essentially lack this vitamin ^[2][7][2,7]. Cobalamin is the only B vitamin not present in plant materials [7]. In veterinary medicine, information on cobalamin metabolism, requirements, and therapy in cats appears to be poor and incomplete.

2. Cobalamin in Cats’ Diet

Cats are obligate carnivores; therefore, they have an essential requirement for nutrients present only in animal tissues [8]. Vitamin B12 is among these essential nutrients because it cannot be synthesized by cats. However, it would appear that a small amount of cobalamin can be synthesized by the intestinal flora present in an area unable to absorb it [2], therefore making the vitamin unavailable. Despite the crucial role of cobalamin, there are currently no studies assessing minimum requirements for weaned kittens, adult cats, or breeding and lactating queens. An amount of 4.5 µg per 1000 Kcal of metabolizable energy (ME), administered through purified diets, was able to maintain normal hemoglobin concentrations in growing kittens [9]. When minimum requirements have not been defined for dietary nutrients, adequate intake can be presumed as the concentration in the diet required to sustain a given life stage [2]. An adequate intake of 4.5 µg per 1000 Kcal ME is suggested, although the NRC recommends including a safety factor of 5.6 µg per 1000 Kcal ME [2]. The European Pet Food Industry Federation (FEDIAF) recommends a dietary cobalamin intake of 4.5 µg per 1000 Kcal ME for breeding kittens and 4.4 µg per 1000 Kcal ME for adult cats [10]. For cat growth, breeding, and adult maintenance, the American Association of Feed Control Officials (AAFCO) recommends a dietary cobalamin intake of 4.5 µg per 1000 Kcal ME [11]. Neither a safe upper limit of cobalamin (the maximum concentration in the diet or an amount that has been associated with adverse effects) or reports of toxicity as a result of cats ingesting high doses are present in the literature [2]. Dietary intake of vitamin B12 is essentially provided by products of animal origin (i.e., meats), which largely represent the principal part of this species’ obligate carnivore diet [8]. Thus, it is unlikely that deficiencies would develop in healthy and well-fed cats. Cobalamin is considered very stable in foodstuffs [2]; however, it is thermolabile. Cooking can inactivate it, depending on the temperature and the type of food ^[12][13][14][12,13,14]. Homemade cooked diets should be supplemented with vitamin B12 accordingly, while raw diets should not be provided unless the recommended intake is achieved. While vitamin B12 content in food for human consumption is well reported [15], it is not well described in pet food. As in other developed economic areas, European pet food companies are not required to declare the analytical or additive value of vitamin B12 on product labels [16]. The dietary source of cobalamin in pet foods has historically been animal products, although most pet food contains bioavailable synthetic cobalamin produced by microbial fermentation. For plant-based diets, appreciated by vegan and vegetarian pet owners, the addition of synthetic cobalamin can meet the animals’ dietary requirement for the vitamin [7].

3. Evaluation of Cobalamin Status and Deficiency in Cats

The serum cobalamin concentration is measured by an automated chemiluminescence competitive binding immunoassay system ^[17][18][19][35,36,37]. The reference interval for feline serum cobalamin concentration is 290–1500 ng/L and values <290 ng/L are considered subnormal ^[17][19][35,37]. A serum cobalamin concentration <100 ng/L represents the lower limit of detection of the chemiluminescent assay system for cats ^[19][20][29,37]. For serum cobalamin assessment, samples should be promptly stored at −20 °C. However, this vitamin remains stable for 5 days if samples are refrigerated at 6 °C even if they are not protected from light ^[21][22][17,38]. The direct influence of diet on serum cobalamin concentration can depend on several factors, and age, sex, and breed play important roles. Older cats frequently show decreased absorption of this vitamin, probably related to a decreased ability to digest it due to reduced ileal and pancreatic function ^[23][24][39,40]. Kittens show lower serum cobalamin concentration during the first months of life; this may be related to the immaturity of the microbiota, the gastrointestinal tract, and the pancreas. A gradual increase in cobalamin serum level is observed in young cats during the first year of age, when microbiota, gastrointestinal tract, and pancreas complete their development ^[25][41]. Male cats usually show significantly higher serum cobalamin concentrations than females ^[23][39]. Higher concentrations of vitamin B12 have been recorded in pure-breed cats than mixed-breed cats ^[26][27][42,43]. Barron et al. (2009) reported that the median serum cobalamin concentration in healthy cats is lower in older and mixed cats, but not in male cats, contrary to what was suggested by Hill et al. (2018) ^[28][44]. The serum cobalamin concentration does not accurately reflect the real availability of cobalamin, because cobalamin-dependent metabolic reactions occur within cells, mainly in the cytoplasm and mitochondria [15]. Methylmalonyl-CoA mutase catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, which is produced by the catabolism of odd-chain fatty acids and amino acids. Succinyl-CoA is a key molecule in the citric acid cycle. A lack of intracellular cobalamin leads to reduced enzyme activity and an intracellular and subsequent systemic accumulation of methylmalonic acid (MMA), resulting in methylmalonic acidemia ^[20][29]. This molecule can also inhibit the activity of carbamoyl phosphate synthetase I, an enzyme of the urea cycle that normally metabolizes ammonia to carbamoyl phosphate. When this metabolic process is impaired, plasma ammonia concentrations typically increase ^[21][17] and neurological disorders can occur ^[29][45]. Therefore, the MMA concentration in cat serum is particularly useful for detecting cobalamin deficiency, as it is directly related to the real bioavailability of the vitamin for intracellular enzymatic reactions ^[30][46]. Serum and urinary MMA concentrations can be determined by gas chromatography–mass spectrometry in fresh or stored samples (−20 °C) ^[17][19][21][17,35,37]. The reference interval of serum MMA concentration defined for cats is 139–897 nmol/L ^[20][29]. The limitation of MMA measurement is mainly the higher cost compared to cobalamin detection [15]. Cats with severely subnormal serum cobalamin concentrations typically show extremely elevated serum MMA concentrations; a 50-fold increase over the highest normal value has been documented ^[30][46]. Although the serum MMA concentration reflects an intracellular cobalamin deficiency, other causes of elevated MMA must be considered, such as renal failure, reduced plasma volume reduction, and abnormalities in hepatic methyl-malonyl CoA mutase activity ^[15][31][15,47]. In both dogs and cats, normal serum MMA concentration with low serum cobalamin status seems to be a possible clinical finding. Low serum cobalamin levels could reflect depletion and redistribution of body reserves prior to real cobalamin deficiency in the mitochondria ^[19][32][33][37,48,49]. There is a lack of available data regarding urinary MMA determination in cats. To reduce the dilution effect and the influence of renal impairment, the urinary MMA-to-creatinine ratio should be considered. In cats, the reference range has been defined as 0.22–0.51 mmol/mol creatinine [3]. While a correlation between serum cobalamin deficiency and increased urinary MMA concentration has been observed in cats, no significant correlation has been detected between serum and urinary MMA concentrations ^[3][34][3,50]. In dogs, measuring urinary MMA concentration appears to be more advantageous due to its high stability in urine; in this species, urinary levels appear to be more concentrated than serum levels (urinary MMA concentration can be up to 40 times higher than serum concentration) ^[21][17]. In humans, total plasma homocysteine levels are used in the diagnosis of cobalamin deficiency. Cobalamin represents an essential cofactor for the enzyme methionine synthase, which is essential for the synthesis of methionine from homocysteine. Cobalamin deficiency blocks this reaction by promoting homocysteine accumulation. When not enough cobalamin is available, the pathway is blocked and homocysteine accumulates within the cells and plasma ^[35][51]. In humans, hyperhomocysteine is related to cobalamin deficiency ^[36][52], whereas in dogs, homocysteine can be used mainly in the diagnosis of familiar cobalamin deficiency specifically in the Chinese Shar-Pei ^[37][53], but only after other diseases have been ruled out ^[38][54]. In cats, homocysteine is not used as a diagnostic marker ^[15][39][15,55], because cobalamin deficiency is not associated with hyperhomocysteinemia in this species ^[19][20][40][29,30,37]. Even when the serum cobalamin concentration is extremely low or undetectable, the homocysteine level will fail to diagnose cobalamin deficiency probably because methionine is commonly added to commercial diets for cats, since it is generally the most limited amino acid in their diet ^[20][29]. A recent study evaluated serum cobalamin concentrations in healthy tigers (Panthera tigris) and tigers affected by pancreatic insufficiency-like syndrome. The results showed much lower B12 concentrations in both healthy and affected tigers than in domestic cats, although reference ranges for serum cobalamin concentrations in tigers have not yet been established ^[41][56]. Another study was conducted on cheetahs (Acinonyx jubatus) to establish reference ranges of species-specific biomarkers of gastrointestinal disease, including cobalamin and MMA. The measured ranges were 470–618 ng/L and 365–450 nmol/L for cobalamin and MMA, respectively, showing that even between closely related species such as the domestic cat and cheetah, there can be differences in gastrointestinal biomarkers ^[42][57].