Regulation of bone cell function by acid-base balance

Bone growth and turnover results from the coordinated activities of two key cell types. Bone matrix is deposited and mineralised by osteoblasts and it is resorbed by osteoclasts, multinucleate cells that excavate pits on bone surfaces. It has been known since the early 20th century that systemic acidosis causes depletion of the skeleton, an effect assumed to result from physico-chemical dissolution of bone mineral. However, our own work has shown that resorption pit formation by cultured osteoclasts was absolutely dependent on extracellular acidification; these cells are inactive at pH levels above about 7.3 and show maximum stimulation at a pH of about 6.9. Bone resorption is most sensitive to changes in H+ concentration at a pH of about 7.1 (which may be close to the interstitial pH in bone). In this region pH shifts of < 0.05 units can cause a doubling or halving of pit formation. In whole-bone cultures, chronic HCO3- acidosis results in similar stimulations of osteoclast-mediated Ca2+ release, with a negligible physico-chemical component. In vivo, severe systemic acidosis (pH change of about -0.05 to -0.20) often results from renal disease; milder chronic acidosis (pH change of about -0.02 to -0.05) can be caused by excessive protein intake, acid feeding, prolonged exercise, ageing, airway diseases or the menopause. Acidosis can also occur locally as a result of inflammation, infection, wounds, tumours or diabetic ischaemia. Cell function, including that of osteoblasts, is normally impaired by acid; the unusual stimulatory effect of acid on osteoclasts may represent a primitive 'fail-safe' that evolved with terrestrial vertebrates to correct systemic acidosis by ensuring release of alkaline bone mineral when the lungs and kidneys are unable to remove sufficient H+ equivalent. The present results suggest that even subtle chronic acidosis could be sufficient to cause appreciable bone loss over time.     

Hijikia fusiforme protects against ovariectomy-induced bone loss in rats

The prophylactic effects of Hijikia fusiforme on bone metabolism were examined using in vitro indices of bone formation and resorption. As the indices of bone formation, osteoblast proliferation and differentiation were measured through mitochondrial enzyme activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] and bone marker alkaline phosphatase (ALP) activity. The aqueous extract of H. fusiforme stimulated the proliferation of the human osteoblast-like cell line MG63 and the ALP activity of the mouse osteoblast-like cell line MC3T3-E1. Moreover, extracellular matrix mineralization was accelerated by the addition of H. fusiforme. As the indices of bone resorption, differentiation of the murine macrophage/osteoclast precursor cell line RAW 264.7 by receptor activator of nuclear factor-κB ligand (RANKL) was measured as tartrate-resistant acid phosphatase-positive multinucleated cells, which were suppressed by H. fusiforme. Additionally, H. fusiforme lowered the secreted amount of RANKL that is required for the osteoclastic differentiation and activation, but the amount of osteoprotegerin as a decoy receptor for RANKL was not affected. The bone-protective effects of H. fusiforme in estrogen-deficient ovariectomized rats were also investigated. Osteoporosis was induced in female Sprague-Dawley rats by ovariectomy for 15 weeks, and then H. fusiforme was orally administered at a dose of 100 mg/kg of body weight every day for 6 weeks. Bone mineral density in the group orally administered H. fusiforme was increased, compared with ovariectomized rats, but not significantly (P>.05). Oral administration of H. fusiforme improved microarchitecture of bone in terms of bone volume (bone volume/total volume ratio) and trabecular separation (P<.05). The amounts of osteocalcin and C-telopeptide type I collagen in serum were measured as the biomarkers for bone formation and resorption. The level of osteocalcin in serum was increased, but not significantly. However, the level of C-telopeptide type I collagen in serum was significantly decreased (P<.05). When the results are taken together, the present study indicates that H. fusiforme might be useful in the treatment of osteoporosis.     

Lycopene I--effect on osteoclasts: lycopene inhibits basal and parathyroid hormone-stimulated osteoclast formation and mineral resorption mediated by reactive oxygen species in rat bone marrow cultures

Osteoclasts have been shown to produce reactive oxygen species (ROS) that can stimulate bone resorption. We explored the hypothesis that lycopene, the antioxidant carotenoid from tomatoes, can inhibit mineral resorption by inhibiting osteoclast formation and the production of ROS. Cells from bone marrow prepared from rat femur were plated into 16-well calcium phosphate-coated Osteologic Multi-test Slides and cultured in alpha-minimal essential medium supplemented with dexamethasone, beta-glycerophosphate, and ascorbic acid. The cells were treated with varying doses of lycopene in the absence or presence of parathyroid hormone (PTH) at the start of culture and at each medium change (i.e., every 48 hours). On day 8, mineral resorption pits were quantitated. Similar, parallel experiments were carried out in 12-well plastic dishes to assess tartrate-resistant acid phosphatase (TRAP) activity. Results showed that lycopene inhibited TRAP + formation of multinucleated cells in both vehicle- and PTH-treated cultures. Osteoclasts reduced nitroblue tetrazolium (NBT) to purple-colored formazan, indicating the presence of ROS in these cells. The formazan-staining cells were decreased by treatment with 10(-5) M lycopene, indicating that lycopene inhibited the formation of ROS-secreting osteoclasts. In conclusion, we have shown that lycopene inhibits basal and PTH-stimulated osteoclastic mineral resorption and formation of TRAP + multinucleated osteoclasts, as well as the ROS produced by osteoclasts. These findings are novel and may be important in the pathogenesis, treatment, and prevention of osteoporosis.     

Nutrition Society Medal lecture. The role of the skeleton in acid-base homeostasis

Nutritional strategies for optimising bone health throughout the life cycle are extremely important, since a dietary approach is more popular amongst osteoporosis sufferers than drug intervention, and long-term drug treatment compliance is relatively poor. As an exogenous factor, nutrition is amenable to change and has relevant public health implications. With the growing increase in life expectancy, hip fractures are predicted to rise dramatically in the next decade, and hence there is an urgent need for the implementation of public health strategies to target prevention of poor skeletal health on a population-wide basis. The role that the skeleton plays in acid-base homeostasis has been gaining increasing prominence in the literature; with theoretical considerations of the role alkaline bone mineral may play in the defence against acidosis dating as far back as the late 19th century. Natural, pathological and experimental states of acid loading and/or acidosis have been associated with hypercalciuria and negative Ca balance and, more recently, the detrimental effects of 'acid' from the diet on bone mineral have been demonstrated. At the cellular level, a reduction in extracellular pH has been shown to have a direct enhancement on osteoclastic activity, with the result of increased resorption pit formation in bone. A number of observational, experimental, clinical and intervention studies over the last decade have suggested a positive link between fruit and vegetable consumption and the skeleton. Further research is required, particularly with regard to the influence of dietary manipulation using alkali-forming foods on fracture prevention. Should the findings prove conclusive, a 'fruit and vegetable' approach to bone health maintenance may provide a very sensible (and natural) alternative therapy for osteoporosis treatment, which is likely to have numerous additional health-related benefits.     

Acid-base imbalance and the skeleton

Summary Humans generally consume a diet that generates metabolic acids leading to a reduction in the concentration of systemic bicarbonate and a fall in pH. In vitro experiments indicate that this metabolic acidosis causes a release of calcium from bone that initially is simply due to physicochemical dissolution of the mineral. On a more chronic basis metabolic acidosis alters bone cell function; there is an increase in osteoclastic bone resorption and a decrease in osteoblastic bone formation. Concomitant with the dissolution and resorption of the bone mineral there is buffering of the addition protons by bone leading to restoration of the systemic pH. Interestingly respiratory acidosis, caused by an increase in the partial pressure of carbon dioxide induces far less bone dissolution and resorption and the additional hydrogen ions are not buffered by bone. As we age we are less able to excrete these metabolic acids due to the normal decline in renal function. We hypothesize that a slight, but significant, metabolic acidosis leads to greater loss of bone mineral and increase potential to fracture. Received: 13 September 2001, Accepted: 20 September 2001     

Animal nutrition and acid-base balance

Summary In domestic animals, acid-base balance may be influenced by nutrition. The major research effort in this area has been made on the prevention of hypocalcemic postparturient paresis in dairy cows. This disorder is caused by the sudden increase of calcium secretion into the colostrum. The manipulation of the dietary cation-anion difference makes it possible to maintain the cows in metabolic acidosis during the critical period that precedes calving, presumably via a mechanism that involves the strong ion difference in the extracellular fluid. As a consequence the mobilization of calcium is enhanced and the incidence of the disorder is decreased. Conversely, a dietary induced metabolic alkalosis leads to a more severe degree of hypocalcemia and the incidence of the disease is increased. The underlying mechanisms of the prevention are only partially understood. Nevertheless, this preventive method is already widely applied in practice. Nutrition effects on acid-base balance also influence growth and food intake in higher vertebrates and fish. As a consequence, the incidence of developmental orthopedic diseases in fast-growing domestic animal species may be affected. Also, the bone mineral content of athletic horses may be influenced by dietary induced modification of the acid-base status. The mineral loss due to metabolic acidosis may lead to an increase in the incidence of stress fractures. This overview should give insight into relevant aspects of nutrition and acid-base balance in domestic animal species. Received: 28 March 2001, Accepted: 10 April 2001     

Experimental evidence for the effects of calcium and vitamin D on bone: a review

Animal models fed low calcium diets demonstrate a negative calcium balance and gross bone loss while the combination of calcium deficiency and oophorectomy enhances overall bone loss. Following oophorectomy the dietary calcium intake required to remain in balance increases some 5 fold, estimated to be approximately 1.3% dietary calcium. In the context of vitamin D and dietary calcium depletion, osteomalacia occurs only when low dietary calcium levels are combined with low vitamin D levels and osteoporosis occurs with either a low level of dietary calcium with adequate vitamin D status or when vitamin D status is low in the presence of adequate dietary calcium intake. Maximum bone architecture and strength is only achieved when an adequate vitamin D status is combined with sufficient dietary calcium to achieve a positive calcium balance. This anabolic effect occurs without a change to intestinal calcium absorption, suggesting dietary calcium and vitamin D have activities in addition to promoting a positive calcium balance. Each of the major bone cell types, osteoblasts, osteoclasts and osteocytes are capable of metabolizing 25 hydroxyvitamin D (25D) to 1,25 dihydroxyvitamin D (1,25D) to elicit biological activities including reduction of bone resorption by osteoclasts and to enhance maturation and mineralization by osteoblasts and osteocytes. Each of these activities is consistent with the actions of adequate circulating levels of 25D observed in vivo.     

Urine pH is an indicator of dietary acid-base load, fruit and vegetables and meat intakes: results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk population study

Evidence exists that a more acidic diet is detrimental to bone health. Although more precise methods exist for measurement of acid-base balance, urine pH reflects acid-base balance and is readily measurable but has not been related to habitual dietary intake in general populations. The present study investigated the relationship between urine pH and dietary acid-base load (potential renal acid load; PRAL) and its contributory food groups (fruit and vegetables, meats, cereal and dairy foods). There were 22,034 men and women aged 39-78 years living in Norfolk (UK) with casual urine samples and dietary intakes from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk FFQ. A sub-study (n 363) compared pH in casual samples and 24 h urine and intakes from a 7 d diary and the FFQ. A more alkaline diet (low PRAL), high fruit and vegetable intake and lower consumption of meat was significantly associated with a more alkaline urine pH before and after adjustment for age, BMI, physical activity and smoking habit and also after excluding for urinary protein, glucose, ketones, diagnosed high blood pressure and diuretic medication. In the sub-study the strongest relationship was found between the 24 h urine and the 7 d diary. In conclusion, a more alkaline diet, higher fruit and vegetable and lower meat intake were related to more alkaline urine with a magnitude similar to intervention studies. As urine pH relates to dietary acid-base load its use to monitor change in consumption of fruit and vegetables, in individuals, warrants further investigation.     

Bone loss induced by dietary magnesium reduction to 10% of the nutrient requirement in rats is associated with increased release of substance P and tumor necrosis factor-alpha

Dietary Mg intake has been linked to osteoporosis. Previous studies have demonstrated that severe Mg deficiency [0.04% of nutrient requirement (NR)] results in osteoporosis in rodent models. We assessed the effects of more moderate dietary Mg restriction (10% of NR) on bone and mineral metabolism over a 6-mo experimental period in rats. At 2, 4 and 6 mo, serum Mg, Ca, parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D, alkaline phosphatase, osteocalcin and urine pyridinoline were measured. Femurs and tibiae were collected for measurement of mineral content, microcomputerized tomography, histomorphometry, and immunocytochemical localization. By 2 mo, profound Mg deficiency had developed as assessed by marked hypomagnesemia and up to a 51% reduction in bone Mg content. These features continued through 6 mo of study. Serum Ca was slightly but significantly higher in Mg-deficient rats than in controls at all time points. At 2 mo, serum PTH was elevated in Mg-deficient rats but was significantly decreased at 6 mo in contrast to control rats in which PTH rose. Serum 1,25-dihydroxy-vitamin D was significantly lower than in controls at 4 and 6 mo. A significant fall in both serum alkaline phosphatase and osteocalcin suggested decreased osteoblast activity. Histomorphometry demonstrated decreased bone volume and trabecular thickness. This was confirmed by microcomputerized tomography analysis, which also showed that trabecular volume, thickness and number were significantly lower in Mg-deficient rats. Increased bone resorption was suggested by an increase in osteoclast number over time compared with controls as well as surface of bone covered by osteoclasts and eroded surface, but there was no difference in osteoblast numbers. The increased bone resorption may be due to an increase in TNF-alpha because immunocytochemical localization of TNF-alpha in osteoclasts was 199% greater than in controls at 2 mo, 75% at 4 mo and 194% at 6 mo. The difference in TNF-alpha may be due to substance P, which was 250% greater than in controls in mononuclear cells at 2 mo and 266% at 4 mo. These data demonstrated that a Mg intake of 10% of NR in rats causes bone loss that may be secondary to the increased release of substance P and TNF-alpha.     

Citrate Supplementation Restores the Impaired Mineralisation Resulting from the Acidic Microenvironment: An In Vitro Study

Chronic metabolic acidosis leads to bone-remodelling disorders based on excessive mineral matrix resorption and inhibition of bone formation, but also affects the homeostasis of citrate, which is an essential player in maintaining the acid–base balance and in driving the mineralisation process. This study aimed to investigate the impact of acidosis on the osteogenic properties of bone-forming cells and the effects of citrate supplementation in restoring the osteogenic features impaired by the acidic milieu. For this purpose, human mesenchymal stromal cells were cultured in an osteogenic medium and the extracellular matrix mineralisation was analysed at the micro- and nano-level, both in neutral and acidic conditions and after treatment with calcium citrate and potassium citrate. The acidic milieu significantly decreased the citrate release and hindered the organisation of the extracellular matrix, but the citrate supplementation increased collagen production and, particularly calcium citrate, promoted the mineralisation process. Moreover, the positive effect of citrate supplementation was observed also in the physiological microenvironment. This in vitro study proves that the mineral matrix organisation is influenced by citrate availability in the microenvironment surrounding bone-forming cells, thus providing a biological basis for using citrate-based supplements in the management of bone-remodelling disorders related to chronic low-grade acidosis.     

Parenteral nutrition-related bone disease.

Parenteral nutrition (PN)-related bone disease remains a problem in patients of all ages. Understanding of the pathogenesis of PN-related bone disease is complicated by the effect of underlying illnesses, therapeutic interventions, and pre-existing nutrition deficiencies before the initiation of PN therapy. Interrelation of various nutrients, for example, calcium, phosphorus, and vitamin D, in their effects on bone mineralization, demands simultaneous assessment of the role of multiple nutrients and increases the difficulty in defining the role of a single nutrient in the development of bone disease. However, recent reports indicate that there exist a number of factors important in the development of PN-related bone disease and some factors such as increased mineral requirement are unique to growing infants whereas other factors such as aluminum toxicity may be common to both adult and pediatric populations. Nonnutritional factors, including chronic use of potent loop diuretics and altered acid-base status, can affect urine mineral loss, cell metabolism, and bone mineralization, particularly in small, preterm infants. Current evidence indicates that the cause of PN-related bone disease is multifactorial, and the prevention of PN-related bone disease awaits better delineation of the exact sequence of pathogenic events.     

Fractures in the elderly and vitamin D

Aging is associated with increased fracture risk. Among the many contributing factors, reduced bone mineral density is highly correlated with this risk. Nutritional factors, i.e. protein, calcium, and vitamin D intake, are essential for maintenance of the skeleton throughout the lifespan and are often compromised in the elderly. Vitamin D is also derived from conversion of precursors in the skin that is stimulated by sunlight. Decreased mobility and avoidance of the sun contribute to vitamin D deficiency in the elderly. The skeletal effects of vitamin D are two-fold: it ensures mineralization of the organic matrix of bone and it mediates mobilization of stored calcium and phosphate from bone to blood to achieve mineral homeostasis. Inadequate levels of vitamin D produce a secondary increase in release of parathyroid hormone that stimulates bone resorption and can result in osteoporosis. If coupled with insufficient intake of calcium, vitamin D deficiency results in osteomalacia. With aging, there is reduced absorption of the vitamin from the diet, reduced conversion to its active metabolites, and resistance to its action in the intestine and in bone. Correction of vitamin D deficiency is an inexpensive task and may require education of clinicians as well as the public. In sum, vitamin D deficiency is prevalent in the elderly and, to correct the adverse skeletal effects, it should be diagnosed, treated, and prevented.     

Effect of potato on acid-base and mineral homeostasis in rats fed a high-sodium chloride diet

Excessive dietary NaCl in association with a paucity of plant foods, major sources of K alkaline salts, is a common feature in Western eating habits which may lead to acid-base disorders and to Ca and Mg wasting. In this context, to evaluate the effects of potato, rich in potassium citrate, on acid-base homeostasis and mineral retention, Wistar rats were fed wheat starch (WS) or cooked potato (CP) diets with a low (0.5 %) or a high (2 %) NaCl content during 3 weeks. The replacement of WS by CP in the diets resulted in a significant urinary alkalinisation (pH from 5.5 to 7.3) parallel to a rise in citrate and K excretion. Urinary Ca and Mg elimination represented respectively 17 and 62 % of the daily absorbed mineral in rats fed the high-salt WS diet compared with 5 and 28 % in rats fed the high-salt CP diet. The total SCFA concentration in the caecum was 3-fold higher in rats fed the CP diets compared with rats fed the WS diets, and it led to a significant rise in Ca and Mg intestinal absorption (Ca from 39 to 56 %; Mg from 37 to 60 %). The present model of low-grade metabolic acidosis indicates that CP may be effective in alkalinising urine, enhancing citrate excretion and ameliorating Ca and Mg balance.     

Vitamin D Interactions with Soy Isoflavones on Bone after Menopause: A Review

Vitamin D is known to increase Ca absorption in adults. However, the threshold vitamin D status to benefit Ca absorption is lower than the target vitamin D status for higher bone mineral density and lower fracture risk, pointing to another pathway for vitamin D to benefit bone. One possibility is by affecting osteoblast and osteoclasts directly. Vitamin D-related bone metabolism may also be affected by soy isoflavones, which selectively bind to the estrogen receptor β and may reduce bone loss in postmenopausal women. We discuss a possible synergistic effect of soy isoflavones and vitamin D on bone by affecting osteoblast and osteoclast formation and activity in postmenopausal women.     

Change in maternal bone mineral density during pregnancy and relationship between the density and foetus growth--a prospective study

In order to study change in maternal bone mineral density during pregnancy and its relationships with bone metabolism and maternal perinatal factors including foetus growth, bone mineral density and bone metabolic markers were measured in 45 pregnant women aged 26-35 years. Specifically, bone mineral density was measured twice, first at 8-20 weeks of gestation and secondly within two weeks postpartum by ultrasonic bone densitometry, while bone metabolic markers were assessed at 8-20 and 26-30 weeks of gestation and at one week postpartum. Bone mineral density and metabolic markers were also examined in 35 non-pregnant controls, twice with a six-month interval. The results of the measurements and subsequent examinations based thereon were as follows: 1. Stiffness as an index of bone density decreased significantly during pregnancy (mean: -4.3%), with wide variation among individuals (-20%-(+)11%). Stiffness in controls did not show any significant change within the 6 months. 2. During pregnancy, bone alkaline phosphatase and osteocalcin levels were significantly lower while urinary hydroxyproline levels (H.P/Cre) were significantly higher than in controls. Significant negative correlations were found between change rate in stiffness and HP/Cre measured at second trimester and postpartum. These results indicate that bone formation is reduced while bone resorption is increased during pregnancy, and that stiffness index reflects the extent of bone resorption. 3. Pre-pregnancy BMI and weight gain during pregnancy could not be linked with change in bone mineral density. 4. The women with greater bone density demonstrated a greater extent of loss postpartum. 5. Postpartum bone mineral density showed a significant, positive correlation with baby birth weight and height. Higher preservation of maternal bone mass is important not only for the mother's health but also for the baby's. Ultrasonic bone densitometry was found to be useful for measuring bone mineral density because it could detect small changes during pregnancy reflecting bone metabolism.     

Biochemical markers of bone remodeling: recent data of their applications in managing postmenopausal osteoporosis

The recent development of new biochemical markers has considerably improved the non invasive exploration of bone turnover. Currently, the most sensitive markers to access bone formation are osteocalcin, bone alkaline phosphatase and N-terminal propeptide of type I procollagen. Blood or urinary immunoassays of pyridinoline, deoxy-pyridinoline and terminal telopeptides type I of collagen are currently the best indices to evaluate the bone resorption. The principal application fields of biochemical markers in postmenopausal osteoporosis, in combination with the measurement of the bone mineral mass, are primarily the monitoring of anti-resorptive therapy response and prediction of bone loss and risk of fractures. In fact, treatment with anti-resorptive drugs is followed by rapid decrease of bone markers levels (3 months for the markers of resorption and 6 months for those of osteo-formation ), whereas the bone mineral density measurements requires at least two years to change significantly.     

小鼠胚胎长骨体外培养的研究

采用自制的旋转装置在国内首次建立了１６天小鼠胚胎长骨体外培养方法，可同时观察新骨的形成、吸收和钙化，为研究骨的生长发育和营养素、毒性物质等其它因素对骨发育的影响提供模型。     

Intake of fruit and vegetables: implications for bone health

'These famous words by Mencken in the early 20th century about the meaning of life and death, may also apply to the struggle of the healthy skeleton against the deleterious effects of retained acid!' (Kraut & Coburn, 1994). The health-related benefit of a high consumption of fruit and vegetables and the influence of this food group on a variety of diseases has been gaining increasing prominence in the literature over a number of years. Of considerable interest to the osteoporosis field is the role that bone plays in acid-base balance. Natural, pathological and experimental states of acid loading and acidosis have been associated with hypercalciuria and negative Ca balance, and more recently the detrimental effects of 'acid' from the diet on bone mineral have been demonstrated. Suprisingly, consideration of the skeleton as a source of 'buffer' contributing to both the preservation of the body's pH and defence of the system against acid-base disorders has been ongoing for over three decades. However, it is only more recently that the possibility of a positive link between a high consumption of fruit and vegetables and indices of bone health has been more fully explored. A number of population-based studies published in the last decade have demonstrated a beneficial effect of fruit and vegetable and K intake on axial and peripheral bone mass and bone metabolism in men and women across the age-ranges. Further support for a positive link between fruit and vegetable intake and bone health can be found in the results of the Dietary Approaches to Stopping Hypertension (DASH) and DASH-Sodium intervention trials. There is now an urgent requirement for the implementation of: (1) fruit and vegetable and alkali administration-bone health intervention trials, including fracture risk as an end point; (2) re-analysis of existing dietary-bone mass and metabolism datasets to look specifically at the impact of dietary 'acidity' on the skeleton.     

Acid-base balance in ruminating calves given sodium hydroxide-treated straw

1.Studies of whole-body balances of non-metabolizable base (NB) and several minerals, and of relevant acid-base quantities in blood and urine, were carried out in two 6-month-old ruminating Holstein X Friesian bull calves fed on fixed rations containing 500 g barley straw/kg diet (group A) to examine the quantitatively important components of the balance of NB and determine the rates of mineral and NB retention associated with normal body growth. 2. Parallel balance studies were conducted in six other bull calves given fixed rations containing 500 g alkali-treated barley straw/kg diet to evaluate the effects of long-term alkali-straw feeding on the rates of body growth and skeletal mineral and NB deposition and the renal control of extracellular electrolyte and acid-base status. The straw component was treated either with 50 g sodium hydroxide/kg dry matter (DM) (group B; two calves), or with 50 g or 100 g NaOH/kg DM and subsequently neutralized with hydrochloric acid (groups C and D, two calves per group). In all groups the animals were given free access to tap water. 3. Throughout the total 105 d experiment, all animals remained healthy and gained weight. Normal body growth group A) was associated with a positive balance of NB (1-2 mmol/kg live weight (LW) per d) due to continuing deposition of dietary NB in 'new tissue', largely in the developing skeleton. 4. During 105 d alkali-straw feeding, the animals showed a remarkable ability to cope with dietary loads of NAOH or sodium chloride, up to about 30 mmol/kg LW per d, without any significant disturbance of extracellular acid-base and electrolyte status or body growth rate. The surplus mineral and NB loads were absorbed and subsequently excreted in an increased volume of urine. Rates of mineral and NB retention were not significantly different from the reference values of group A and remained within the range of values reported from similar studies. In all groups, maintenance of normal whole blood and plasma acid-base and electrolyte status was accounted for by efficient renal control of the composition of the extracellular fluid compartment.