Thiamine, like most hydrophilic micronutrients, is absorbed most effectively in the mucosal cells of the upper jejunum and to a lesser extent from the duodenum and ileum (69). Additionally, dietary proteins are digested in the digestive tract, releasing thiamine (70). Thiamine absorption occurs by a dual mechanism that is saturable at low physiological concentrations (oral intake <5 mg) and diffusive at higher concentrations (39). The human intestinal absorption of thiamine is regulated by the two high-affinity carriers: human thiamine transporters-1 (hTHTR-1), encoded by Solute Carrier Family 19 Member 2 (SLC19A2), and hTHTR-2, encoded by SLC19A3 (71–73). SLC19A2 mutations cause thiamine-responsive megaloblastic anemia (TRMA), which is accompanied by hyperglycemia and sensorineural deafness (74). The hTHTR-1 facilitates the transport of thiamine from the intestine to the portal circulation via the basolateral membrane and thus enters erythrocytes (42, 75). These cells contain about 80% of thiamine in the form of TPP while very low concentrations are present in plasma (76). Thiamine concentrations are highest in the heart (0.28–0.79 mg/100 g), followed by kidneys (0.24–0.58 mg/100 g), liver (0.20–0.76 mg/100 g), and brain (0.14–0.44 mg/100 g), with brain concentrations lasting the longest (36). The half-life of thiamine is only 9.5–18.5 days while the human body does not retain thiamine at concentrations greater than 30 mg (37, 42). The low reservoir of thiamine, combined with its short half-life and continuous demand for utilization in metabolic activities, necessitates steady thiamine consumption (77). With marginal intake, the symptoms of thiamine deficiency may appear within 72 h (78). The reuptake of thiamine from urine is facilitated by hTHTR1 and hTHTR2 in the basolateral membrane and brush border membrane and renal tubular cells. Long-term diuretic use has been linked to thiamine deficiency. Loop diuretics, such as furosemide, cause large thiamine loss, up to 2 times the baseline (79). Thiamine is also eliminated through feces, sweat and breast milk (41, 75, 80). Thiamine levels in breast milk are normally in the range of 0.14–0.21 mg/L, although they vary depending on the diet (81).

There are two principal methods of evaluating the thiamine status: assessment of the degree of ThDP saturation of a thiamine-dependent enzyme [erythrocyte transketolase (ETK) assay] and evaluation of the metabolites of thiamine inaccessible tissues (81). The erythrocyte transketolase (ETK) assay is the widely used, most comprehensive method of determining thiamine status as it reveals the functionality of the vitamin (23). Patients with an increase in ETKA of more than 25% are at high risk of TD, those with increases of 16–25% are at moderate risk of TD, and those with activity coefficients of less than 15% have adequate thiamine status (37, 82). Direct assessment of ThDP in whole blood by High-Performance Liquid Chromatography (HPLC) is the method of choice with high precision, sensitivity and specificity (83, 84). Since 80% of the total thiamine content of whole blood is present as active ThDP in red blood cells making this method superior and more reflective of body stores of thiamine. For normal healthy individuals, the expected concentration of ThDP in whole blood is 70-180 nmol/L (82, 85). The metabolites of thiamine can also be determined by HPLC embedded with ultraviolet-visible spectroscopy (UV/VIS) or fluorescence detectors and Liquid chromatography-mass spectrometry (LC-MS) (86).

Since the human body does not store thiamine for long, an adequate daily supply is required to maintain homeostasis. The recommended nutrient intake (RNI) is the daily intake, which meets the nutrient requirements of almost all healthy individuals in an age- and sex-specific population group (87). RNI of thiamine is 1.2 mg/day and 1.1 mg/day for men and women respectively. During pregnancy and lactation, the requirement increases to 1.4 and 1.5 mg/day respectively. The intake for infants is set at 0.2 mg/day (0–6 months) and 0.3 mg/day (7–12 months) (81). In addition to this, the thiamine requirement is increased after the consumption of a high carbohydrate diet; for every 2,000 kcal consumed daily, the minimum thiamine requirement is set at 0.66 mg (88).

The initial signs of thiamine deficiency are vague that might easily be linked to a variety of illnesses. The presenting symptoms include unrelenting fatigue, mood changes, muscle pain, decreased tendon reflexes, subtle impairment in memory, anorexia, tachycardia, and tachypnoea (144–146). Various reports suggest that initial symptoms like GI disturbance (nausea, vomiting, discomfort, and dysmotility) may be the primary signs of thiamine deficiency (144, 147). The GI symptoms of thiamine deficiency are often confused with pregnancy-related nausea and vomiting, making the early diagnosis difficult.

Cardiomyocytes require a steady supply of energy, and thiamine insufficiency causes disruptions in aerobic respiration pathways, interfering with proper heart function (148). The involvement of the cardiovascular system may take two forms: chronic form or acute fulminant form (Shoshin beriberi). The chronic form comprises of three phases. The first phase is marked by peripheral vasodilation resulting in a high-cardiac output state, wide pulse pressure, tachycardia, sweating, and warm skin. This results in salt and water retention, regulated by the renin-angiotensin-aldosterone system of the kidneys. In the second phase, as the vasodilation progresses further, the kidneys sense a relative reduction of volume and adapt by retaining salt resulting in edema. The third phase is characterized by considerable edema which subjects the heart to a very high workload in order to produce the requisite cardiac output. This causes an overuse injury to cardiomyocytes, resulting in tachycardia, edema, warm extremities, liver and splenic congestion and elevated arterial and venous pressures which are chiefly manifested as chest pain (149). During pregnancy, the most common cardiac manifestations associated with thiamine deficiency include progressive dyspnoea of NYHA Class 2 to 3, generalized edema, and marked right ventricular dilation (133). Additionally, pericardial effusion, intraventricular septum and mild mitral insufficiency have also been reported (150).

The acute fulminant form of wet beriberi is known as “Shoshin beriberi” in which the vasodilation continues causing acute heart failure, cardiogenic shock, and lactic acidosis (142, 151). In severe deficiency, echocardiography shows dilated ventricles (particularly the right ventricle, which is commonly associated with pulmonary hypertension), a D-shaped left ventricle, and a drastically reduced ejection fraction (152). An immediate response to therapy is noted following intravenous infusion of thiamine (153).

Since thiamine has a crucial physiological role in energy metabolism, deficiency leads to a decrease in oxidative metabolism. Biochemical derangements disrupt the supply of ATP to neurons, causing oxidative stress, and decreased synthesis of neurotransmitters [e.g., acetylcholine, glutamate, aspartate, and gamma amino butyric acid (GABA)] which produces a toxic neuroexcitatory state (154, 155). Impairment of the pentose phosphate pathway results in decreased nerve conduction velocity because thiamine participates in the maintenance of myelin sheaths (156). The primary symptoms of dry beriberi include lower limb, bilateral and symmetric, paresthesia, calf muscle tenderness, burning of feet and plantar dysesthesia. The absence of ankle jerk reflexes can determine peripheral neuropathy. Persistent deficiency results in the loss of tendon reflexes, atrophy of the calf and thigh muscles, and, lastly, foot drop, and toe drop (35). Dry beriberi in pregnancy can initially present as poor visual acuity, diplopia, and gait ataxia along with bilateral horizontal nystagmus (157).

Wernicke’s encephalopathy is an acute neuropsychiatric disorder caused by thiamine deficiency that is frequently coupled with alcohol misuse because of poor diet, carbohydrate-rich diet, inhibition of intestinal ATPase by alcohol, and depletion of magnesium (158). The deficiency of thiamine in WE is considered to cause apoptotic cell death due to N-methyl-D-aspartate (NMDA) toxicity, resulting in neurological symptoms (159). The classical triad of WE includes nystagmus and ophthalmoplegia, changes in mental status, and gait ataxia (160). The mental changes are mostly the result of thalamic or mammillary body involvement and vary from disorientation to mental sluggishness, apathy, poor awareness of the present situation, difficulty concentrating, and, if untreated, coma, and death (161). Classically, WE occur in alcoholics, however, HIV-infected patients, pregnant women suffering from hyperemesis gravidarum, and postoperative bariatric patients are also at risk (162–164). WE in pregnancy and lactation has been reported widely with symptoms varying from mild horizontal nystagmus, dysarthria, and right hemiparesis to more severe neurological manifestations like edematous splenium of the corpus callosum (165) with ataxia being the commonest manifestation (166). A report suggests memory loss as the presenting feature and bilateral lateral rectus palsy as the most common form of ophthalmoparesis in WE (167).

Korsakoff psychosis is a severe form of WE characterized by amnesia, confusion, and confabulation with little or no working memory (168). It occurs in patients, who have previously had WE but did not receive prompt and appropriate thiamine replacement therapy (169). Because of their close relationship, these two disorders are frequently referred to as Wernicke–Korsakoff syndrome (170). Pregnant women are especially at risk of developing Wernicke–Korsakoff syndrome due to hyperemesis gravidarum (171). Hyperemesis gravidarum remains the main risk factor for the development of any complication associated with pregnancy and lactation (172). Wernicke–Korsakoff syndrome typically develops following malnutrition in patients with prolonged self-neglect, alcohol abuse, and other substance abuse (173). Thiamine deficiency occurs in alcoholics because alcohol is calorie-dense yet nutrient-poor. Furthermore, diarrhea and the low capacity of the liver to store vitamins and the impaired conversion of thiamine to the active compound TPP in alcoholics and substance abusers results in impaired storage of thiamine (174). In addition, Korsakoff’s syndrome in alcoholics may be caused by the build-up of lesions during recurrent subclinical episodes of thiamine shortage (175, 176), with ethanol neurotoxicity possibly playing a role (177, 178). Treatment of Wernicke’s encephalopathy with appropriate doses of parenteral thiamine may avoid the development of Korsakoff’s syndrome if administered promptly. According to the guidelines of the Royal College of Physicians, alcoholic individuals at risk for developing WE may be treated with an intravenous infusion of 500 mg of thiamine hydrochloride in 100 mL of normal saline over the course of 30 minutes, thrice daily for 2–3 days. This must be followed by intramuscular dosing of 250 mg of thiamine daily for 3–5 days (173, 179–181).

Donnino (144), reported an unusual, under-recognized form of a gastrointestinal syndrome associated with thiamine deficiency characterized by anorexia, nausea, vomiting, abdominal discomfort, and lactic acidosis, for the first time which came to be known as gastric beriberi (144, 182). However, some consider it to be the prodrome of a more sinister form of thiamine deficiency i.e. WE or that it could be the effect of the thiamine deficient state itself (147). It is well recognized that insufficiency of thiamine causes decreased ATP production in the liver raising the liver enzymes and leading to gastrointestinal symptoms. Our team have also reported various manifestations of gastric beriberi in the otherwise well-fed population of Kashmir (183). Also, vomiting alone has been linked to lower activity of erythrocyte transketolase (33). In addition, to the already known risk factors for beriberi, gastric beriberi may be more common in gastric resection (for malignancy), bariatric surgery, total gastrectomy, Billroth II gastrectomy, or Roux-en-Y diversion (184).

Due to rapid growth, high energy demand, and a high metabolic rate, the thiamine requirements in infancy are high relative to body size. This places newborns at the highest risk of thiamine deficiency in the first year of life (135). In the first months of life, new-borns are more vulnerable to the effects of thiamine deficiency (Figure 3), and exclusively breastfed infants of thiamine-deficient mothers are at the greatest danger (17), which can result in death within hours of clinical presentation if left untreated (26). Infantile beriberi can present as vomiting, refusal to breastfeed, irritability, and constant loud crying which, in some cases, progresses to a “silent cry,” or aphonia (185). If at this stage thiamine is not administered, it can progress into signs and symptoms of congestive heart failure, like tachypnoea, tachycardia, pulmonary edema, hepatomegaly, and, occasionally, cyanosis occurs; at this point, clinical deterioration is generally rapid (108, 186). Many reports suggest that mechanical rice milling coupled with dietary taboos during the postpartum period and formula feeding leads to acute life-threatening metabolic acidosis and pulmonary hypertension in exclusively breastfed infants (187–191). Children who survive thiamine-deficiency-related WE continue to have developmental problems such as impairments in syntax, morphology, reading, phonological working memory, lexical abilities, epilepsy, motor function loss, and intellectual disability (192, 193).

SLC19A2 is a thiamine transporter gene found on the membrane of numerous human tissues, including that of the gastrointestinal tract and encoding the hTHTR1, which aids in the transfer of thiamine across the cell membrane (198). Homozygous, heterozygous or missense mutations in SLC19A2 cause an autosomal recessive condition known as thiamine-responsive megaloblastic anemia (TRMA) or thiamine metabolism dysfunction syndrome 1 or Roger’s syndrome (OMIM 249270) (199, 200). This condition primarily occurs in populations with consanguineous partners and is characterized by a clinical core triad of megaloblastic anemia, non-autoimmune diabetes mellitus, and early-onset sensorineural deafness (201). Cardinal features manifest in infancy or adolescence (202, 203). Other manifestations include cardiac abnormalities, optic atrophy, retinal abnormalities, stroke or epileptic-like episodes and short stature (204, 205). High-dose thiamine supplementation has been shown to delay the onset of diabetes, and generate immediate hematopoietic response but does not affect sensorineural hearing loss in children (200, 206, 207).

Mutations in SLC19A3 (encoding hTHTR2) cause a neurometabolic autosomal recessive disorder known as thiamine metabolism dysfunction syndrome type 2 or biotin-responsive basal ganglia disease (BBGD) or biotin-thiamine responsive basal ganglia disease (BTBGD; OMIM: 607483). This was first described in the Arabian population with a high degree of parental consanguinity and a common missense mutation, p.thr422ala (208). Depending upon the age of onset, this disease is categorized as classical childhood BBGD, early-infantile Leigh-like syndrome/atypical infantile spasms and adult Wernicke’s-like encephalopathy (209). The disease is characterized by acute and recurring episodes of encephalopathy (often triggered by febrile illness, trauma, vaccines), dystonia, rigidity, confusion, difficulties with speech and swallowing, dysarthria, external ophthalmoplegia and seizures, in association with symmetrically distributed brain lesions in the caudate nuclei, putamen, medial thalami and, less frequently, cerebral cortex, brainstem and cerebellum (210–212). Early treatment with biotin (5–10 mg/kg/day) in combination with thiamine (100–900 mg/day) effectively reverses the disease (213, 214).

SLC25A19 encodes mitochondrial thiamine transporter and so far three distinct missense mutations have been discovered affecting this gene (215). The two mutations, G125S and S194P, collectively cause an autosomal recessive disease known as thiamine metabolism dysfunction syndrome-4 [(THMD4) (Bilateral striatal degeneration and progressive polyneuropathy type) (OMIM 613710)] and is characterized by childhood episodic encephalopathy, often associated with a febrile illness, causing transient neurologic dysfunction and a slowly progressive axonal polyneuropathy (216). A third homozygous mutation, G177A, causes a severe autosomal recessive disorder called Amish lethal microcephaly (MCPHA; OMIM 607196). This disease is characterized by microcephaly apparent at birth, markedly delayed psychomotor development, brain abnormalities, and episodic encephalopathy associated with lactic acidosis and -ketoglutaric aciduria (217).

TPK1 converts free thiamine to TPP, as such any mutation in the TPK1 sequence prevents the phosphorylation of thiamine to an active isomer. The onset of the disease is usually early childhood with clinical features of acute encephalopathic episodes, elevated serum, and CSF lactate, learning difficulties, progressive motor dysfunction (e.g., gait abnormalities, ataxia, dystonia, and spasticity) in the striatal, basal ganglia, and cerebellar parts of the brain, while cognition appeared to be unaffected. An early diagnosis is warranted as the condition largely remains treatable, however, diagnosis is delayed due to clinical overlap with other metabolic diseases, including Leigh syndrome (196). The treatment chiefly depends upon oral thiamine supplementation (100–200; 500 mg/day) (201, 218) or thiamine in combination with niacin, biotin, α-lipoic acid, and ketogenic diet (219, 220).

Most of the reports of thiamine deficiency in Sub-Saharan Africa have been occasional, involving groups of refugees, soldiers, prisoners, and a few distant communities (223–227). A recent study by Bourassa and colleagues assessed the thiamine status of women of reproductive age living in rural Gambia and the association between thiamine status and seasonality. The study reported that around 35.8% of the sample was at high risk of thiamine deficiency (ETKac ≥ 1.25) and the risk of thiamine deficiency was significantly higher in the wet (47.9%) compared with the dry season (22.9%) (P < 0.001) (30). The reliance on rice remains an important risk factor among Gambian people, especially women, and the cases peak in the rainy season when food supplies are lowest and there is intense agricultural activity with increased energy expenditure (228).

Thiamine deficiency disorders and suboptimal thiamine status have recently been identified in Cambodia (135). A case-control study conducted by Coats et al. evaluated the whole blood ThDP levels in 27 infants diagnosed with beriberi and their mothers and matched them with healthy Cambodian and American controls. Surprisingly, there was no significant difference in whole blood ThDP levels between Cambodian mothers and infants with and without beriberi but both pairs of Cambodian women and infants had much lower ThDP levels than the American controls (229). Another cross-sectional study was conducted by Whitfield et al. to assess the status of thiamine in women of childbearing age in rural and urban Cambodia, compared with women in Canada. The study reported that thiamine deficiency (ThDP ≤ 90 nmol/L) was common among both urban (39%) and rural (59%) Cambodian women (P < 0.001), whereas <20% of Vancouver women were thiamine deficient (P < 0.001) (136). A similar study by Whitfield et al. among a nationally representative sample of Cambodian women of childbearing age (15 ± 49 years) and their young children (6 ± 69 months) found that women had a lower mean (95% CI) ThDP of 150 nmol/L (146 ± 153) compared to children, 174 nmol/L (171 ± 179; P < 0.001). Using different cut-off values for ThDP, 27–78% of women and 15–58% of children were thiamine deficient, which was especially prevalent among infants aged 6 ± 12 months (135). In addition, various studies have reported cases of infantile beriberi from different areas of Cambodia (34, 186, 230).

A study by McGready et al. (29) among the postpartum Karen refugee population has reported that at 3 months postpartum, thiamine deficiency, reflected by ETKA ≥ 1.20% was found in 57.7% of mothers, 26.9% of whom had severe deficiency (ETKA > 1.25%). A number of studies report childhood thiamine deficiency and the high infant mortality attributed to the deficiency of thiamine (26, 231, 232). A study by Stuetz et al. evaluated the ThDP in whole blood and thiamine in the breast milk of lactating women. It was found that the prevalence of women with low whole blood ThDP (<65 nmol/L) was 5% and with deficient breast-milk total thiamine (<300 nmol/L) was 4% (233). Further, thiamine deficiency was also reported among children with malaria, parasitic infection and acute diarrhea (27, 234, 235). A recent study reported an outbreak of peripheral neuropathy in Bueng Kan Provincial Prison in northeast Thailand associated with thiamine deficiency (236).

Thiamine deficiency has primarily been reported from Northeast India, especially Kashmir, with some scattered cases from South India (221, 237). A number of reports of infantile beriberi have been reported from North India primarily due to postpartum customary dietary restrictions (16, 238–240). A number of case reports have documented WE in women with hyperemesis gravidarum which underlines the significance of thiamine supplementation for pregnant women experiencing protracted vomiting (134, 166, 241–245). Thiamine-responsive acute pulmonary hypertension of early infancy and acute life-threatening metabolic acidosis in exclusively breast-fed infants has been reported intermittently from North India, especially Kashmir (188–190, 246). Another recent study from Kashmir has reported thiamine-responsive neuropathy among peripartum women (28). A case series by Hegde et al. (133) reported the neurological and cardio-pulmonary manifestations of thiamine deficiency in pregnancy and lactation. A case report from the Siaha district of Mizoram, India bordering Myanmar reported that mothers taking the kuhva (raw areca nut) daily during the last pregnancy were independently associated with higher infant mortality (247).

Several case series of infantile beriberi and suboptimal thiamine status among children have been reported from Laos. The assessment of a cohort of 778 sick infants (0–12 months) found that 13.4% have thiamine deficiency and are defined as basal ETK activity < 0.59 (33). A large-scale cross-sectional survey of 22 villages in Luang Namtha Province found that of 468 live-born infants, 50 (10.6%, 95% CI: 8.0–13.8) died during the first year. According to verbal autopsies, 17 deaths (3.6%) were caused by suspected infantile thiamine deficiency. Of 127 mothers, 60 (47.2%) reported edema and paraesthesia; while among 127 infants, 2 (1.6%) had probable thiamine deficiency, and 8 (6.8%) had possible thiamine deficiency (32).

Thiamine deficiency has been reported among infants from resource-poor settings of Myanmar, like refugee camps, and displaced communities (248). It is the second most common cause of infant deaths between 28 days and 1 year of age in Myanmar (249). A nationally representative survey of infant mortality reported that 0.3% of live births died from TDDs before 5 years of age (23, 250).

In 2005, several infants fed on thiamine-deficient soy infant formula were admitted to several intensive care units in Israel with encephalopathy and were later diagnosed as suffering from thiamine deficiency (251, 252). Pediatric thiamine deficiency was perceived as being eradicated or anecdotal in high-income countries, however, from 2000–2020, 389 cases of pediatric thiamine deficiency have been documented with various predisposing factors including, genetic causes, lifestyle (diabetes, obesity, and excessive consumption of sweetened beverages), eating disorders, cancer, gastrointestinal disorders/surgeries, critical illness, and artificial nutrition (253). A case report of a 10-week-old girl with infantile beriberi from Reunion Island, a French Island, has recently been documented (254).

Incorporation of a wide range of thiamine-rich foods like legumes (pulses, beans and groundnuts), lentils etc. in the food basket is a very resourceful approach in improving the thiamine status of the deficient population. Nutrition education along with diversification of food should be encouraged by making vegetable gardening a regular component of all disaster-affected population relief programmes. In places where white-milled rice is a staple food, parboiled undermilled rice should be promoted as a thiamine-rich alternative (255, 256).

Changing dietary patterns to promote the consumption of thiamine-rich foods while decreasing the consumption of known thiamine antagonists and thiamine-poor foods may improve thiamine intake. The two major approaches to dietary modification include the reduction of losses of thiamine during the preparation and cooking of the meal and the reduction of the intake of anti-thiamine factors. The loss of thiamine during preparation is minimized by reducing the number of washes of the rice before cooking and cooking rice in two volumes of water only (257, 258). Cooking losses of thiamine in vegetables can be decreased by washing vegetables before chopping them into small pieces, keeping cooking time to a minimum, consuming freshly prepared meals immediately, and cooking vegetables in a small amount of water and consuming the water (259).

It is well-documented that anti-thiamine factors including, thiamine antagonists and thiaminases contribute to the development of TDDs, especially in populations with marginal thiamine intake (260). Nutrition education and awareness programmes help ameliorate thiamine deficiency by lowering the intake of anti-thiamine components and improving thiamine nutriture.

Food fortification is defined as the practice of deliberately increasing the content of essential micronutrients in food to improve the nutritional quality of the food supply and to provide a public health benefit with minimal risk to health (261). Food fortification is a low-cost, tenable technique for preventing micronutrient deficits (137). Large-scale food fortification of centrally processed food vehicles has a long history of use in industrialized countries for the successful reduction of micronutrient deficiencies and improvement of health outcomes, such as reduced odds for anemia, cretinism, or neural tube defects by providing iron, iodine, or folic acid, respectively (262, 263). Fortification aims to raise the target population’s thiamine consumption to a level consistent with minimal risk of TDDs. Primarily, two thiamine salts are used for fortification: thiamine hydrochloride for liquid vehicles and thiamine mononitrate for dry preparations, however, both are heat labile and destabilized by humidity, light, sulfites and oxygen (264). The vehicle for thiamine fortification must be consumed by the general population throughout the year, must be cost-effective and stable, and should primarily be processed at a centralized facility (265). The most common vehicles for thiamine fortification include rice, wheat flour, corn meal, fish and soy sauces, and salt (81) while sugar, vegetable oil, bouillon cubes, and curry powders are less commonly used vehicles (266). In addition, research is ongoing on biofortification which involves using traditional plant breeding methods, agronomic approaches, or genetic engineering to improve a plant’s micronutrient content (267, 268).

Supplementation refers to the administration of relatively substantial amounts of micronutrients, typically in the form of pills, capsules, or syrups. It has the benefit of being able to provide an appropriate amount of a particular nutrient in a highly absorbable form, and it is frequently the quickest option to control deficiency in people or population groups considered deficient (261). In emergencies, including famines, embargos, refugee settings, etc., thiamine supplementation is the mainstay for averting worse clinical outcomes of thiamine deficiency. Typically, procurement and purchase of micronutrients in somewhat costly pre-packaged form is cost-ineffective and requires high patient compliance. However, as a preventive measure, thiamine tablets can be supplied to pregnant and lactating mothers through prenatal and postnatal clinics to prevent infantile beriberi, especially in exclusively breastfed infants (269).