The study design is a prospective observational study undertaken at the Lagos University Teaching Hospital which ran from April to December 20008 after institutional ethics review board committee approval.
Two cohorts of children were investigated. The first sets of cohort (cohort A) were five children who had been placed on calcium supplementation and vitamin D (cholecalciferol) in appropriate dosage for six months without an appreciable improvement in their biochemical and clinical status. The second set of cohorts (cohort B) were fifty two consecutively presenting children with rickets, who were older than 2 years referred to the paediatrics endocrine outpatient for limb deformity. Any child that was included in the study conformed to the inclusion and exclusion criteria as stated below.
Clinical Features for diagnosis of rickets
Subject must have limb deformities with evidence of rachitic changes in the epiphyses, such as widening and knobby prominence of the wrist and ankle joints and rachitic rosary. These are common clinical features seen in Nigerian children with rickets according to findings by Thatcher et al [5, 7] and Oduwole et al .
Apart from the above criteria, subjects that are included into cohort A must also not be responding to calcium supplementation and cholecalciferol therapy at six months assessment of clinical and biochemical features post institution of therapy. Such a subject was deemed not responding to therapy.
Physical examination was performed by the principal investigator for all the children.
Biochemical Indices for diagnosis of rickets
Subject must have the following biochemical parameters, low serum calcium, serum phosphorus which may be low, or normal and high serum alkaline phosphatase. Interpretation of biochemical values were based on the laboratory normal range values for age. The laboratory normal ranges were serum calcium 2.2-2.8 mmol/L, serum phosphate 0.7-1.3 mmol/L and serum alkaline phosphatase 40-90 U/L. All laboratory indices were done in the tertiary laboratory using their quality control method.
Subject in cohort A must also at six months assessment of biochemical features post institution of therapy have continuous lowering of serum calcium and phosphate levels and increasing level of plasma alkaline phosphatase.
Radiological Indices for diagnosis of rickets
The radiological survey of the limbs must show evidence of changes in the epiphyses such as osteopenia, widening of growth plate, decrease radio-density at sub-zone of provisional ossification.
Subject in cohort A, must also show depreciation in their radiological skeletal survey after being on calcium supplementation and cholecalciferol therapy for minimum of six months.
The radiology reports were made by the same senior radiologist to provide standardization, prevent error and bias.
Any subject who had the following was excluded. These are hemoglobinopathy, urinary tract infection, evidence of hypokalemia or sodium retention to rule out hypoaldosteronism. Other exclusion criteria were severe protein malnutrition, liver disease, chronic diarrhea or malabsorbtion, abnormal serum creatinine level and any child whose caretaker consent could not be obtained. Appropriate laboratory investigations were done to rule out these parameters. Children on drugs that could be associated with rickets like phenobarbitone were also excluded. Any child below the age of 2 years was excluded because of the long hours of the test.
A total of seven subjects were excluded for either non participation in the ammonium chloride test or lack of parental consent. One was excluded from cohort a group and six from the cohort B group. The remaining 50 children studied filled a preformatted tested questionnaire to obtain biodata, type of housing, average daily period of exposure to sunlight, three days food diary, drug history and parents level of education and income to classify into social economic class using the Olusanya et al criteria .
Urine calcium Measurement
24 hours urine calcium evaluation was done for each subject. The children were ambulatory and on a free diet. Prior to the day of the ammonium chloride test the caretakers or parent were advised to discard the first urine passed on waking up on the day of collection, subsequently all urine passed that day and that passed first thing on the day of test was collected into a specific container and brought to the test area. Urine collected were not exposed to sunlight or kept in a hot environment. Where a refrigerator was available, urine were kept in it or in a cool place in a black bag. Volume of urine collected was measured and then evaluated for its urine calcium concentration. Calciuria level in relation to the body weight have been found to be relatively constant in children except for a decrease during puberty . Using the body weight, subject was deemed hypercalciuric if 24 hours urine calcium was more than 1.0 mmol/kg/day (4 mg/kg/day) [15, 16]. This cut off point was used by Manz et al  during their study of 24 hour urinary calcium in healthy British and African children and it was found dependable.
The short time ammonium chloride test
The subjects performed the modified short term ammonium chloride loading test after conforming to the inclusion and exclusion criteria and properly filling the preformatted form. The short term ammonium chloride test which is the modified form of the loading test by Wong and Davies  lasting for eight hours was chosen because it has been found to be sensitive and tolerated by children[18–20].
The children collected their urine for 24 hours prior to coming for the loading test as stated above. On arrival at the testing room, child was made comfortable. The subjects' last meal was at least eight hours prior to test. Test commenced from 7 am and ended at 1 pm. On arrival fresh urine and blood samples were collected for baseline urine pH and serum bicarbonate. This was followed by ingestion of ammonium chloride solution calculated at 0.1 gm/kg and dissolved in 100 mls of water. Ammonium chloride is a colorless and odorless type of salt that should make the blood slightly more acidic when introduced. Thereafter urine was collected every 2 hours over the next eight hours.
Urine pH was determined immediately after voiding or collection to prevent loss of carbon dioxide into the atmosphere. Blood samples were collected twice during the study, at the beginning of test and 3 hours after ingestion of solution. The blood samples were analyzed within a maximum of the hour of collection to prevent loss of CO2 which will give an incorrect (lower) value of bicarbonate. Electrolytes namely sodium, chloride and potassium were also measured. This was to calculate the anion gap thereby differentiating the type of acidosis present in the subject. The urine pH was analyzed in duplicate using the pH meter Orion Research Ionalyser/model 399A, Cambridge, Mass, USA, after standardization with different known buffer solutions provided with the pH meter. A child was said to be unable to acidify the urine if the pH remained above 5.5 after eight hours of ingestion of ammonium chloride solution. The normal serum bicarbonate concentration range is 22-30 mmol/L. In cases of tubular acidosis bicarbonate lower than 20 mmol/L may signify acidosis though mild and must be without any abnormal anion gap . Therefore a fall of serum bicarbonate to 18- 20 mmol/L without any abnormal anion gap was used as a cut off point for classification of mild acidosis.
There were two treatment modalities.
Modality 1 for those without idRTA
Subjects were placed on 5000 I.U of vitamin D in Arachis oil (calciferol) daily orally and one tablet of effervescence CaC1000 by Sandoz to give 600 mg of elemental calcium daily. Dietary counseling was given on calcium rich foods and calcium fortified foods or drinks such as fish bone, milk, egg, butter/margarine, cocoa drinks to augment the supplement from CaC 1000 by Sandoz.
Modality 2 for those with idRTA
Shohl's solution was added for alkalization of the urine to modality 1. Shohl's solution is a palatable solution, which is a combination of sodium citrate and citric acid. It is readily metabolized into bicarbonate and gives an equivalent of 1 ml = 1 mmol of bicarbonate. It was given at a dose of 2 mmol per kilogram of body weight per day in three divided doses for easy timing and compliance by subjects. It is eliminated via urine with less than 5% unchanged. Shohl's solution was continued for all subjects with idRTA after healing had been achieved. It has minimal side effect such has nausea, vomiting, stomach pain and water retention from sodium in the sodium citrate .
Any subject in cohort A without idRTA would be managed as a case of vitamin D resistant rickets.
Subjects were seen at six weeks interval in the outpatient. On presentation subjects were examined by the investigator for changes in clinical features of rickets, any sign of sodium retention such as rapid weight gain, blood pressure measurement. Blood was taken to monitor changes in the biochemical indices. Radiological survey was repeated after six months of therapy modality. The values of the pre and post introduction of Shohl's solution to the therapy parameters were compared. Any improvement or otherwise was noted.
Statistical analysis was done using the Microsoft excels software. Results are expressed as mean (SD), (%), z score, student t-test, 95% CI, probability (p <0.01).
The study protocol was approved by the ethical review committee at Lagos University Teaching Hospital and consent forms were signed by the caretakers of the subjects. There were no conflicting interests.