Table 1.

Evidence table, N-balance studies

Reference	Study design	Population	Outcome measures	Intervention/exposure	Dietary assessment method	No of subjects analysed	Intervention	Results	Study quality and relevance, Comments A-C
Campbell et al., 2001 (8) USA	Controlled metabolic study	10 healthy men and postmenopausal women aged 55–77 y	N-balance and body composition (whole body density, body water, mid-thigh muscle area)	Diet was a rotating cycle of three daily menus of lacto-ovo-vegetarian foods (animal protein from egg and dairy)	14 week strictly controlled dietary intake trial. Weeks 2, 8 and 14 at the clinic for testing and evaluating: four consecutive 24-h urine and four-day fecal collections. No assessment of miscellaneous losses CT scans of the dominant leg at week 2 and 14. The remainder of the study was conducted on an outpatient basis	Four men and six women	Eucaloric diets containing 0.8 g protein/kg body weight (BW) /day	Steady state reached at week 2. Unchanged BW and body composition, but between week 2 and 14 decreased urinary nitrogen excretion and decreased mid-thigh muscle area	B No young control group, few participants, only one dose of protein. The RDA of 0.8 g protein/kg BW/day may be marginally inadequate and may result in loss of skeletal muscle
Campbell et al., 2008 (9) USA	Controlled metabolic study	Healthy volunteers, young (n=27) 29–30 ±7–8 y and old (n=21) 72–75 ±6–4 y, Young vs. old and men vs. women	N-balance	Low protein diet: 0.5 g /kg BW Medium protein diet: 0.75 g/kg BW High protein diet: 1.0 g /kg BW. Animal based egg and dairy protein	Strict dietary control. All weekday morning meals were consumed at the research center, lunch, dinner and weekend meals were packaged and taken home. Duplicate portions of all foods and beverages on days 7–10 of each trial. Stool collections on day 7–9 of each trial. 24-h urine collections on day 7–10 and 14–17 of each trial. Miscellaneous losses assumed to be 5 mg nitrogen/kg BW/d	Young n=23 Old n=19	Three 18-d periods, minimum one week habitual diet between the periods. 3-d rotation of menus Eucaloric diet (estimated energy intake from estimated REE times PAL). Morning meal consumed at dining facility, lunch, dinner and weekend meals were packaged and taken home	BW unchanged. N-balance not different between the four groups. Estimated requirement expressed pr. kg BW was not significantly different for the young vs. old or men vs. women. Mean protein requirement was lower for older women vs. older men, but expressed pr. kg FFM there was no significant difference. For all subjects combined the adequate protein allowance was estimated to be 0.85 ±0.21 g/kg BW/d	A The estimated adequate protein allowance was 0.85 ±0.21 g/kg BW/d and not significantly different from Rand et al., (3)
Morse et al., 2001 (10) USA	Controlled metabolic study	12 healthy women aged 70–81 y	N-balance, body composition, resting metabolic rate	Low protein diet: 0.5g /kg BW Medium protein diet: 0.75 g/kg BW High protein diet: 1.0 g /kg BW. Animal based egg and dairy protein. A basal menu of solid foods (0.40g/kg BW) supplemented with a protein mixture of cheese and protein powder	Strict dietary control. Duplicate portions of all foods and beverages, stool collections and 24-h urine collections on day 7–10 and 14–17 of each trial. Miscellaneous losses assumed to be 8 mg nitrogen/kg BW/d	11 women	Three 18-d periods, minimum one week habitual diet between the periods. 3-d rotation of menus. Day 1: a eucaloric very low protein diet (0.18 g/kg BW) used to enhance adaption. Morning meal consumed at dining facility, lunch, dinner and weekend meals were packaged and taken home	Unchanged BW and body composition, Mean adequate protein allowance was estimated to be 0.90 g/kg BW at week 2 and 0.76 g/kg BW at week 3, but the urinary nitrogen excretion decreased between week 2 and 3 indicating that a steady state was not reached yet.	B No power calculation. No young control group. Women only. Short-term studies (< 2 weeks) may be inadequate to achieve N-balance. The 11 women are also included in the study from 2008 by Campbell et al (9)
Rand et al., 2003 (3)	Meta-analysis	19 balance studies among healthy persons	N-balance		Controlled nitrogen (protein) intake. Measured urine and faeces, correction for dermal and miscellaneous losses. At least 3 test protein intakes, given for 10–14 days, urinary and faucal excretion data for, the last 5 d, eucaloric diet studies and a adaption period ≈5 d.	N=235		The median estimated protein requirement of good quality protein: 0.66 g/kg BW/d and the estimated RDA: 0.83 g/kg BW/d No differences for adult age groups, sex or protein source	B Not sufficient power to detect possible differences between e.g. sex and age groups. Only one study with elderly available in the analysis. Data suggest a possible age difference in nitrogen utilization that needs to be further explored
Walrand et al., 2009 (11) USA	Controlled study Single-blinded	10 healthy young 24 ±1 y (5 men) and 10 old 70 ±2 y (5 men) volunteers	N-balance, glomerular filtration rate (GFR), Muscle synthesis	High protein (HP): 3.0 g/kg FFM and usual protein (UP): 1.5 g/kg fat free mass (FFM)	All food was prepared at the metabolic ward. 24-h urine collection at the end of each 10-d trial. No stool collections and no estimate for miscellaneous losses.	10 young and nine old	10 days of HP (Young: 2.08±0.07 g/kg BW and old: 1.79±0.1 g/kg BW) or UP (young: 1.04±0.03 g/kg BW, and old 0.89±0.05 g/kg BW) protein diets in a cross-over design, separated by 2–8 weeks	Unchanged body weight. N-balance and muscle protein synthesis did not differ with age. GFR was lower in older participants and they had a lesser increase in GFR during the HP diet corresponding to 77% of younger people at the UP and 58% of younger people during HP	B Short-term, but relevant because of the high protein intake. No age related difference in N-balance but concern about an HP diet corresponding to ca. 24E% in the elderly because of potential adverse effect on the kidney function